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`as) United States
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`a2) Patent Application Publication 10) Pub. No.: US 2006/0217311 Al
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`Dix et al. Sep. 28, 2006 (43) Pub. Date:
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`US 20060217311A1
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`(54) VEGF ANTAGONIST FORMULATIONS
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`Related U.S. Application Data
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`(76)
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`Inventors: Daniel Dix, LaGrangeville, NY (US);
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`Kelly Frye, Pomona, NY (US); Susan
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`Kautz, Albany, NY (US)
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`(60) Provisional application No. 60/665,125, filed on Mar.
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`25, 2005.
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`Publication Classification
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`Correspondence Address:
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`REGENERON PHARMACEUTICALS, INC
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`777 OLD SAW MILL RIVER ROAD
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`TARRYTOWN, NY 10591 (US)
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`(21) Appl. No.:
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`11/387,256
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`(22)
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`Filed:
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`Mar. 22, 2006
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`Int. CL
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`(2006.01)
`AGIK 38/17
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`(2006.01)
`AGIK 31/7012
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`(2006.01)
`AGIK 31/4172
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`creccccccccsercecrssseeee 514/12: 514/53; 514/400
`(52) US. CD.
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`ABSTRACT
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`Formulations of a vascular endothelial growth factor
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`(VEGF)-specific fusion protein antagonist are provided
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`including a pre-lyophilized formulation, a reconstituted lyo-
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`philized formulation, and a stable liquid formulation. Pref-
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`erably, the fusion protein has the sequence of SEQ ID NO:4.
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`CELLTRION - EXHIBIT 1033
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`CELLTRION - EXHIBIT 1033
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`US 2006/0217311 Al
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`VEGF ANTAGONIST FORMULATIONS
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`CROSS-REFERENCE TO RELATED
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`APPLICATIONS
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`[0001] This application claims the benefit under 35 USC §
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`119(c) of U.S. Provisional 60/665,125 filed 25 Mar. 2005,
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`which application is herein specifically incorporated by
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`reference in its entirety.
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`BACKGROUND OF INVENTION
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`[0002]
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`1. Field of the Invention
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`[0003] The present inventionis directed to pharmaceutical
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`formulations comprising agents capable of inhibiting vas-
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`cular endothelial growth factor (WEGF), and to methods for
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`making andusing such formulations. The invention includes
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`pharmaceutical formulations having increased stability.
`2. Statement of Related Art
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`[0004]
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`[0005] Vascular endothelial growth factor (VEGF) expres-
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`sion is nearly ubiquitous in human cancer, consistent with ils
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`role as a key mediator of tumor neoangiogenesis. Blockade
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`of VEGF function, by binding to the molecule or its
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`VEGFR-2 receptor, inhibits growth of implanted tumorcells
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`in multiple different xenograft models (see, for example,
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`Gerberet al. (2000) Cancer Res. 60:6253-6258). A soluble
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`VEGF-specific fusion protein antagonist, termed a “VEGF
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`trap” has been described (Kimet al. (2002) Proc. Natl. Acad.
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`Sci. USA 99:11399-404; Holash et al. (2002) Proc. Natl.
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`Acad. Sci. USA 99:11393-8), which applications are spe-
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`cifically incorporated by reference in their entirety.
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`[0006] Lyophilization (freeze drying under controlled con-
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`ditions) is commonlyused for long term storage of proteins.
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`The lyophilized protein is substantially resistant to degra-
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`dation, aggregation, oxidation, and other degenerative pro-
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`cesses while in the freeze-dried state (see, for example, U.S.
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`Pat. No. 6,436,897).
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`BRILT SUMMARY OP TIE INVENTION
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`[0007]
`Stable formulations of a VEGF-specific fusion
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`protein antagonist are herein provided. The pharmaceuti-
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`cally acceptable formulations of the invention comprise the
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`VEGF“trap” antagonist with a pharmaceutically acceptable
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`carrier. In specific embodiments, liquid and freeze-dried, or
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`lyophilized formulations are provided.
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`the invention features a stable
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`[0008]
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`liquid formulation of a VEGF-specific fusion protein
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`antagonist, comprising a fusion protein comprising, a recep-
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`tor componentconsisting essentially of an immunoglobulin-
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`like (Ig) domain 2 of a first VEGF receptor and Ig domain
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`3 of a second VEGF receptor, and a multimerizing compo-
`nent, one or more buffers, and one or more thermalstabi-
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`lizers. In a specific embodiment of the VEGF-specific fusion
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`protein antagonist, the first VEGF receptor is Flt] and the
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`second VEGFreceptor is Flk1 or Flt4. In a more specific
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`embodimentthe fusion protein has the amino acid sequence
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`of SEQ ID NO:2 or SEQ ID NO:4. In one embodiment, the
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`buffer is a phosphate buffer and/or citrate. More preferably,
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`the buffers are phosphate andcitrate. In one embodiment, the
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`thermal stabilizers are NaCl and/or sucrose. More prefer-
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`ably, the thermal stabilizers are both NaCl and sucrose.
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`Ina specific embodiment, the stable liquid formu-
`[0009]
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`lation of a VEGF-specific fusion protein antagonist com-
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`prises 1-10 mM phosphate buffer, 1-10 mM citrate, 25-150
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`mM NaCl, 5-30% sucrose, 10-50 mg/ml of the fusion
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`protein, at a pH of about 6-6.5. In a more specific embodi-
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`ment, the stable liquid formulation comprises 5 mM phos-
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`phate buffer, 5 mM citrate buffer, 100 mM NaCl, 20%
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`sucrose, 25 mg/mlof the fusion protein, at a pH of about 6.0.
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`Additionally, polysorbate may be present, for example 0.05-
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`0.15%polysorbate 20. The stable liquid formulation of the
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`VEGF-specific fusion protein antagonist of the invention
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`exhibits little or no precipitation after storage of a 25 mg/ml
`VEGFformulation for about 6 months at -80° C.and little
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`or no precipitation after storage for 6 months at 5° C.
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`[0010]
`Ina second aspect, the invention features a high
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`concentration stable liquid formulation of a VEGFantago-
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`nist comprising 1-50 mM histidine, 25-150 mM NaCl,
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`5-30% sucrose, 50-100 mg/mlof the fusion protein, at a pH
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`of about 6-6.5, and either 0.1-0.5%polysorbate or 1-5%
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`PEG.In a morespecific embodiment,the high concentration
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`stable liquid formulation comprises 10 mM histidine, 50
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`mM NaCl, 5-20% sucrose, 50-100 mg/ml of the fusion
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`protein, at a pH of about 6.0-6.5, with either 0.1% polysor-
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`bate (c.g., polysorbate 20) or 3% PEG (c.g., PEG 3350). The
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`high concentration stable liquid formulation of the VEGF-
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`specific fusion protein antagonist of the invention cxhibits
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`less than about 3% degradation after 15 months ofstorage at
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`5° C. (75 or 100 mg/ml VEGFtrap protein) or less than
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`about 1.5% degradation after 24 months (50 mg/ml).
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`[0011]
`the invention features a pre-
`In a third aspect,
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`lyophilized formulation of a vascular endothelial growth
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`factor (VEGF)-specific fusion protein antagonist, compris-
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`ing a (i) fusion protein comprising a receptor component
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`consisting essentially of an immunoglobulin-like ([g)
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`domain 2 of a first VEGF receptor and Ig domain 3 of a
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`second VEGF receptor, and a multimerizing component,(i1)
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`a buffer, (111) an organic co-solvent or bulking agent, and(iv)
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`one or more lyoprotectants. In various embodiments, the
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`buffer is histidine, the organic co-solvent or bulking agentis
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`PEG,and the lyoprotectant(s) is at least one of glycine and
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`sucrose. In one embodiment, the pre-lyophilized. formula-
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`tion of the invention does not contain a preservative.
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`In one embodiment of the pre-lyophilized formu-
`[0012]
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`lation of the invention, the formulation comprises 5-50 mM
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`histidine, 0.1-3.0% PEG, 0.25-3.0% glycine, 0.5-6.0%
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`sucrose, and 5-75 my/ml of the fusion protein, al a pH of
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`about 6.0-6.5. In any embodiment, the pre-lyophilized for-
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`mulation may further comprise up to 0.05 mM citrate and/or
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`0.003-0.005% polysorbate. The polysorbate present maybe,
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`for example, polysorbate 20.
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`[0013]
`Ina more specific embodiment, the pre-lyophilized
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`formulation comprises about 10 mM histidine, about 1.5%
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`PEG 3350, about 0.75% glycine, about 2.5% sucrose, and
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`about 12.5 to 75 mg/ml VEGF-specific fusion protein, at a
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`pH of about 6.25.
`In specific embodiments,
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`protein comprises the protein sequence of SEQ ID NO:4,
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`present as a multimer, e.g., a dimer. In separate embodi-
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`ments, the reconstituted formulation is 2 times the concen-
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`tration of the pre-lyophilized formulation, e.g., a 20 mg
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`fusion protein/ml! pre-lyophilized formulation is reconsti-
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`tuted to a final formulation of 60 mg fusion protein/ml.
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`Generally, the lyophilized formulation is reconstituted with
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`US 2006/0217311 Al
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`Sep. 28, 2006
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`sterile water suitable for injection. In one embodiment, the
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`reconstitution liquid may be bacteriostatic water.
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`[0014]
`In a preferred embodiment,
`the pre-lyophilized
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`formulation consists essentially of about 10 mM histidine,
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`about 1.5% PEG 3350, about 0.75% glycine, about 2.5%
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`sucrose, and about 50 mg/mlofthe fusion protein having the
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`sequence of SEQ ID NO:4 as a dimer, at a pH of about 6.25.
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`Citrate (less than or equal to about 0.02 mM) and/or polysor-
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`bate (less than or equal to about 0.0005%) may be present.
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`Optionally, the pre-lyophilized formulation does not contain
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`a preservative, a phosphate buffer, and/or more than trace
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`amounts of NaCl. In one embodiment, the pre-lyophilized
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`formulation consists of about 10 mM histidine, about 1.5%
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`PEG 3350, about 0.75% glycine, about 2.5% sucrose, and
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`about 50 mg/ml of the VEGFtrap protein (SEQ ID NO:4),
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`pH 6.3, and upon reconstitution contains 20 mM histidine,
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`3% PEG, 1.5% glycine, about 5% sucrose, and about 100
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`mg/ml VEGFtrap protein.
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`[0015]
`Ina fourth aspect, the invention features a method
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`of producing a lyophilized formulation of a VEGF-specific
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`fusion protein antagonist, comprising subjecting the pre-
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`lyophilized formulation of the invention to lyophilization to
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`generate a lyophilized formulation. The lyophilized formu-
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`lation may be lyophilized by any method knownin the art
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`for lyophilizing a liquid.
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`[0016]
`In a fitth related aspect, the invention features a
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`method ofproducing a reconstituted lyophilized formulation
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`of a VIGI'-specific fusion protein antagonist, comprising
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`reconstituting the lyophilized formulation ofthe invention to
`a reconstituted formulation. In one embodiment, the recon-
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`stituted formulation is twice the concentration of the pre-
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`lyophilized formulation, e.g., the method of the invention
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`comprises: (a) producing a pre-lyophilized formulation of a
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`VEGF-specific fusion protein antagonist, (b) subjecting the
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`pre-lyophilized formulation of step (a) to lyophilization; and
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`(c) reconstituting the lyophilized formulation of step (b).
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`In specific embodiments of the method ofproduc-
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`ing a reconstituted lyophilized formulation, a pre-lyo-
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`philized solution is present in a vial as a 25 mg VEGE-
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`specific fusion protcim antagonist per ml solution of pre-
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`formulation, which
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`reconstituted to an 50 mg/ml solution. In another embodi-
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`ment, a 30 mg/m] pre-lyophilized solution is lyophilized and
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`reconstituted to a 60 mg/ml solution. In another embodi-
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`ment, a 40 mg/mlpre-lyophilized solution is lyophilized and
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`reconstituted to a 80 mg/ml solution. In another embodi-
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`ment, a 12.5 mg/ml pre-lyophilized solution is lyophilized
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`and reconstituted to a 25 mg/ml solution. In another embodi-
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`ment, a 50 mg/ml pre-lyophilized solution is lyophilized and
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`reconstituted to a 100 mg/ml solution. In another embodi-
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`ment, a 75 mg/ml pre-lyophilized solutionis lyophilized and
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`reconstituted to a 150 mg/ml solution. Preferably, the recon-
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`stituted lyophilized formulation does not contain a preser-
`valive.
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`[0018] Other objects and advantages will become apparent
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`from a review of the ensuing detailed description.
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`DETAILED DESCRIPTION OF THE
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`INVENTION
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`[0019] The present invention is not limited to particular
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`methods, and experimental conditions described, as such
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`methods and conditions mayvary.It is also to be understood
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`the terminology used herein is for the purpose of
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`describing particular embodimentsonly, and is not intended
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`to be limiting unless indicated, since the scope of the present
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`invention will be limited only by the appended claims.
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`[0020] As used in this specification and the appended
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`claims, the singular forms “a”, “an”, and “the”include plural
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`references unless the context clearly dictates otherwise.
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`Thus for example, references to “a method” include one or
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`more methods, and/or steps of the type described herein
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`and/or which will become apparent to those persons skilled
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`in the art upon reading this disclosure.
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`[0021] Unless stated otherwise, all technical and scientific
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`terms and phrases used herein have the same meaning as
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`commonly understood by one of ordinary skill in the art to
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`which the invention belongs. Although any methods and
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`materials similar or equivalent to those described herein can
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`be usedin the practice or testing of the present invention, the
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`preferred methods and materials are now described. All
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`publications mentioned herein are incorporated herein by
`reference.
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`General Description
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`[0022]
`Safe handling and administration of formulations
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`comprising proteins represent significant challenges to phar-
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`maceutical formulators. Proteins possess unique chemical
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`and physical properties that present stability problems: a
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`variety of degradation pathways exist for proteins, impli-
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`cating both chemical and physical
`instability. Chemical
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`instability includes deamination, aggregation, clipping of
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`the peptide backbone, and oxidation of methionineresidues.
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`Physical instability encompasses many phenomena, includ-
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`ing, for example, aggregation.
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`[0023] Chemical and physical stability can be promoted
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`by removing water from the protein. Lyophilization (freeze-
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`drying under controlled conditions) is commonly used for
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`long-term storage of proteins. The lyophilized protein is
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`substantially resistant to degradation, aggregation, oxida-
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`tion, and other degenerative processes while in the freeze-
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`dried state. The lyophilized protein is normally reconstituted
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`with water optionally containing a bacteriostatic preserva-
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`tive (c.g., benzyl alcohol) prior to administration.
`Definitions
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`[0024]
`‘The term “carricr’” includes a dilucnt, adjuvant,
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`excipient, or vehicle with which a composition is adminis-
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`tered. Carriers can include sterile liquids, such as,
`for
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`example, water andoils, including oils of petroleum, animal,
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`vegetable or synthetic origin, such as, for example, peanut
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`oil, soybean oil, mineral oil, sesame oil and thelike.
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`[0025] The term “excipient” includes a non-therapeutic
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`agent added to a pharmaceutical composition to provide a
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`desired consistency or stabilizing effect. Suitable pharma-
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`ceutical excipients include, for example, starch, glucose,
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`lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel,
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`sodium stearate, glycerol monostearate, talc, sodium chlo-
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`ride, dried skim milk, glycerol, propylene, glycol, water,
`ethanol and thelike.
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`[0026] The term “lyophilized”or “freeze-dried” includes a
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`state of a substance that has been subjected to a drying
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`procedure such as lyophilization, where at
`least 50% of
`moisture has been removed.
`
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`

`

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`US 2006/0217311 Al
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`Sep. 28, 2006
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`[0027] The phrase “bulking agent” includes a compound
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`that is pharmaceutically acceptable and that adds bulk to a
`
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`lyo cake. Generally, acceptable bulking agents known to the
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`art include, for example, carbohydrates,
`including simple
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`sugars such as dextrose,ribose, fructose andthelike, alcohol
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`sugars such as mannitol, inositol and sorbitol, disaccharides
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`includingtrehalose, sucrose and lactose, naturally occurring
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`polymers such as starch, dextrans, chitosan, hyaluronate,
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`proteins (e.g., gelatin and serum albumin), glycogen, and
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`synthetic monomers and polymers. In the formulations of
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`the invention, PEG 3350 is an organic co-solvent which is
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`used to stabilize the fusion protein when agitated, mixed, or
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`handled, and as a bulking agent to help produce an accept-
`able bulk.
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`[0028] The term “lyoprotectant” includes a substance that
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`may be addedto a freeze-dried or lyophilized formulation to
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`help maintain protein structure when freeze-dried or lyo-
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`philized.
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`[0029] A “preservative” includes a bacteriostatic, bacte-
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`riocidal, fungistatic or fungicides compound that is gener-
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`ally added to formulations to retard or climinate growth of
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`bacteria or other contaminating microorganisms in the for-
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`mulations. Preservatives include, for example, benzy] alco-
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`hol, phenol, benzalkonium chloride, m-cresol, thimerosol,
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`chlorobutanol, methylparaben, propylparaben and the like.
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`Other examples of pharmaceutically acceptable preserva-
`tives can be found in the USP.
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`VEGF Antagonists
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`[0030] An VEGF antagonist is a compound capable of
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`blocking or inhibiting the biological action of vascular
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`endothelial growth factor (VEGF), and includes fusion pro-
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`teins capable of trapping VEGF. In a preferred embodiment,
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`the VEGFantagonist is the fusion protein of SEQ ID NO:2
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`or 4; more preferably, SEQ ID NO:4. In specific embodi-
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`ments, the VEGF antagonist is expressed in a mammalian
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`cell line such as a CHO cell and may be modified post-
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`translationally. In a specific embodiment, the fusion protein
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`comprises amino acids 27-457 of SEQ ID NO:4 andis
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`glycosylated at Asn residues 62, 94, 149, 222 and 308.
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`[0031] The VEGF antagonist of the methods and formu-
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`lations of the invention can be prepared by any suitable
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`method knownin the art, or that comes to be known. The
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`VEGFantagonist is preferably substantially free of protein
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`contaminants at the time it is used to prepare the pharma-
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`ceutically acceptable formulation. By “substantially free of
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`protein contaminants” is meant, preferably, that at least 90%
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`of the weight of protein of the VEGF-specific fusion protein
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`antagonist preparation used for making a formulation is
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`VEGFfusion protein antagonist protein, more preferably at
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`least 95%, most preferably at least 99%. The fusion protein
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`is preferably substantiallyfree of aggregates. “Substantially
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`free of aggregates” means that at least 90% of the weight of
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`fusion protein is not present in an aggregate at the time the
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`fusion protein is used to prepare the pharmaceutically effec-
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`tive formulation. The fusion protein of the methods and
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`formulations of the invention may contain low or trace
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`amounts of compounds as a results of the purification
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`process, for example, lowor trace amounts ofcitrate and/or
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`polysorbate. In one embodimentof the pre-lyophilized for-
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`
`
`mulation of the invention containing about 50 mgof fusion
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`protein/ml, citrate may be present at a concentration of about
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`0.02 mM and/or polysorbate may be present at a concen-
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`tration of about 0.0005%. If the pre-lyophilized formulation
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`is reconstituted after lyophilization to half of the original
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`volume(e.g., 100 mg/ml of fusion protein), the resulting
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`concentrations may be 0.04 mM citrate and/or 0.001%
`
`polysorbate.
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`Lyophilization and Lyophilized Formulations
`
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`[0032]
`In one aspect ofthe invention, a pharmaceutically
`
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`
`
`acceptable formulation comprising a VEGI -specific fusion
`
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`
`
`protein antagonist is provided, wherein the formulation is a
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`freeze-dried or lyophilized formulation. Lyophilized formu-
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`lations can be reconstituted into solutions, suspensions,
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`
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`emulsions, or any other suitable form for administration or
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`use. I yophilized formulations are typically first prepared as
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`liquids, then frozen and lyophilized. The total liquid volume
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`before lyophilization can be less, equal to, or more than, the
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`final reconstituted volume of the lyophilized formulation.
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`The lyophilization process is well knowntoa those of ordi-
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`
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`nary skill in the art, and typically includes sublimation of
`water from a frozen formulation under controlled condi-
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`tions.
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`[0033] Lyophilized formulations can be stored at a wide
`
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`range of temperatures. Lyophilized formulations may be
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`stored below 25° C., for example, refrigerated at 4° C., or at
`
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`room temperature (e.g., approximately 25° C.). Preferably,
`
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`lyophilized formulations are stored below about 25° C.,
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`morepreferably, at about 4-20° C.; below about 4° C.; below
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`about -20° C.; about —40° C.; about -70° C., or about -80°
`Cc.
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`[0034] Lyophilized formulations are typically reconsti-
`
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`
`
`tuted for use by addition of an aqueoussolution to dissolve
`
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`
`
`the lyophilized formulation. A wide variety of aqueous
`
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`
`
`solutions can be used to reconstitute a lyophilized formula-
`
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`
`
`tion. Preferably, lyophilized formulations are reconstituted
`
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`using water. Lyophilized formulations are preferably recon-
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`stituted with a solution consisting essentially of water (e.g.,
`
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`USP WEI, or water for injection) or bacteriostatic water
`
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`(e.g., USP WFI with 0.9% benzyl] alcohol). However, solu-
`
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`tions comprising buffers and/or excipients and/or one or
`
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`more pharmacetically acceptable carries can also be used.
`
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`[0035] Freeze-dried or lyophilized formulations are typi-
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`cally prepared from liquids, that is, from solutions, suspen-
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`sions, emulsions, and the like. Thus, the liquid that is ta
`
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`undergo freeze-drying or lyophilization preferably com-
`
`
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`
`prises all components desired in a final reconstituted liquid
`
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`
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`formulation. As a resull, when reconstituted, the freeze-dried
`
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`or lyophilized formulation will render a desired liquid
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`
`
`formulation upon reconstitution. A preferred liquid formu-
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`
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`
`
`lation used to generate a freeze-dried or lyophilized formu-
`
`
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`
`
`
`lation comprises a VEGF-specific fusion protein antagonist
`
`
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`
`
`
`in a pharmaceuticallyeffective amount, a buffer, a stabilizer,
`
`
`
`
`
`
`and a bulking agent. Freeze-dried or lyophilized formula-
`
`
`
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`
`
`
`
`tions preferably comprise histidine, since histidine, in com-
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`
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`
`
`
`
`parison to phosphate,
`is more effective at stabilizing the
`
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`
`
`
`fusion protein when the fusion protein is lyophilized.
`
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`
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`
`
`
`Organic cosolvents, such as PEG 3350, are used to stabilize
`
`
`
`
`
`
`
`
`the fusion protein when agitated, mixed, or handled. A
`
`
`
`
`
`
`
`lyoprotectant
`is preferably used in freeze-dried or lyo-
`
`
`
`
`
`
`philized formulations. Lyoprotectants help to maintain the
`
`
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`
`
`
`
`
`secondary structure of proteins when freeze-dried or lyo-
`
`
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`
`
`
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`philized. Two preferred example lyoprotectants are glycine
`
`
`
`
`
`
`
`and sucrose, which are preferably used together.
`
`

`

`
`
`US 2006/0217311 Al
`
`
`
`Sep. 28, 2006
`
`
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`
`
`
`
`
`
`Stable Liquid Formulations
`
`
`
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`
`
`[0036]
`the invention provides a stable
`In one aspect,
`
`
`
`
`pharmaceutically acceptable formulation comprising a
`
`
`
`
`
`
`
`VEGE-specific fusion protein antagonist, wherein the for-
`
`
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`
`
`
`mulation is a liquid formulation. Preferably,
`the liquid
`
`
`
`
`
`formulation comprises a pharmaceutically effective amount
`
`
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`
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`
`
`
`of the fusion protein. The formulation can also comprise one
`
`
`
`
`
`
`
`or more pharmaceutically acceptable carriers, buffers, bulk-
`
`
`
`
`
`
`ing agents, stabilizers, preservatives, and/or excipients. An
`
`
`
`
`
`example of a pharmaceutically acceptable liquid formula-
`
`
`
`
`
`
`tion comprises a VEGF -specific fusion protein antagonist in
`
`
`
`
`
`
`a pharmaceutically effective amount, a buffer, a co-solvent,
`and one or morestabilizers.
`
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`
`[0037] A preferred liquid formulation comprises phos-
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`
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`phate buffer, an organic co-solvent, and one or more thermal
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`stabilizers to minimize formation of aggregates and low
`
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`
`
`molecular weight products when stored, and about 10 mg/ml
`
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`
`
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`to about 50 mg/ml fusion protein, wherein the formulation
`
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`
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`is from about pH 6.0-6.5. A preferred liquid formulation
`
`
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`
`
`comprises about 5 mM phosphate buffer, about 5 mM
`
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`
`
`citrate, about 100 mM NaCl, about 25% sucrose, and about
`
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`
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`10-50 mg/ml fusion protein, wherein the formulation is at a
`
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`
`
`pH ofabout6.0; optionally polysorbate maybepresent(c.g.,
`
`
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`
`
`
`
`
`0.1%polysorbate 20), Although cither NaCl or sucrose can
`
`
`
`
`
`
`
`be used as a stabilizer, a combination of NaCl and sucrose
`
`
`
`
`
`
`
`
`has been established to stabilize the fusion protein more
`
`
`
`
`
`
`effectively than either individual stabilizer alone.
`
`
`
`
`
`
`[0038]
`Stability is determined in a number of ways at
`
`
`
`
`
`
`
`specified time points, including determination of pH, visual
`
`
`
`
`
`
`inspection of color and appearance, determination of total
`
`
`
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`
`
`
`
`
`protein content by methods known in the art, e.g.. UV
`
`
`
`
`
`spectroscopy, SDS-PAGE,size-exclusion HPLC, bioassay
`
`
`
`
`
`
`determination ofactivity, isoelectric focusing, and isoaspar-
`
`
`
`
`
`
`
`tate quantification. In one example of a bioassay useful for
`
`
`
`
`
`
`determining VEGF antagonist activity, a BAF/3 VEGFR1/
`
`
`
`
`
`
`EPORcell line is used to determine VEGF 165 binding by
`
`
`
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`
`
`
`
`
`the VEGF-specific fusion protein antagonist of the inven-
`tion.
`
`
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`
`
`
`
`[0039] Formulations, whether liquid or freeze-dried and
`
`
`
`
`
`
`lyophilized, can be stored in an oxygen-deprived environ-
`
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`
`
`
`ment. Oxygen-deprived environments can be generated by
`
`
`
`
`
`
`
`
`
`
`storing the formulations under an mert gas such as, for
`
`
`
`
`
`example, argon, nitrogen, or helium.
`EXAMPLES
`
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`
`
`[0040] Before the present methodsare described,it is to be
`
`
`
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`
`
`
`understood that this invention is not limited to particular
`
`
`
`
`
`
`
`methods, and experimental conditions described, as such
`
`
`
`
`
`
`
`methods and conditions may vary.It is also to be understood
`
`
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`
`
`
`
`
`
`the terminology used herein is for the purpose of
`that
`
`
`
`
`
`
`
`describing particular embodiments only, and is not intended
`
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`
`
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`to be limiting, since the scope of the present invention will
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`be limited only to the appended claims.
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`[0041] As used in this specification and the appended
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`claims, the singular forms “a”, “an”, and “the” include plural
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`references unless the context clearly dictates otherwise.
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`Thus for example, a reference to “a method”includes one or
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`more methods, and/or steps of the type described. herein
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`and/or which will become apparent to thase persons skilled
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`in the art upon reading, this disclosure and so forth.
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`[0042] Unless defined otherwise, all technical and scien-
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`tific terms used herein have the same meaning as commonly
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`understood by one ofordinary skill in the art to which this
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`invention belongs. Although any methods and materials
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`similar or equivalent to those described herein can be used
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`in the practice or testing of the present
`invention,
`the
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`preferred methods and materials are now described. All
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`publications mentioned herein are incorporated herein by
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`reference in their entirety.
` Example 1
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`Stability of a 50 mg/ml Liquid Formulation of
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`VEGF Trap
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`[0043] A liquid formulation containing, 10 mM phosphate,
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`50 mM NaCl, 0.1% polysorbate 20, 20% sucrose, and 50
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`mg/ml VEGF trap (SEQ ID NO:4), pH 6.25, wasstored at
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`5° C. and samples tested at 3, 6, 9, 12, 18 and 24 months.
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`Stability was determined by SE-HPLC.Theresulls, shown
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`in Table 1, showthat 98.6% and 98.3%ofVEGFtrap protein
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`remain undegraded at 12 and 24 months, respectively.
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`Turbidity was measured at OD,,, nm; and percent recovered
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`protein bysize exclusion HPLC.
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`TABLE1
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`Stability of 50 mg/ml VEGF Trap Protein When
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`Stored at_5° C.
`(VGFT-SS065
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`Visual
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`Months Appearance
`
`0
`Pass
`3
`Pass
`6
`Pass
`9
`Pass
`12
`Pass
`18
`Pass
`24
`Pass
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`Turbidity
`0.00
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`0.00
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`0.01
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`0.01
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`0.01
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`0.00
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`0.00
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`pH
`6.2
`6.2
`6.2
`63
`6.3
`6.3
`6.2
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`% VEGF Trap
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`Recovered
`

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