I, Frank Reginald David Apps, hereby certify that the following document is, to the best
`of my knowledge and belief, a true and accurate translation from Japanese into English.
`
`PCT Publication No. WO00/51629 (Sato)
`
` I
`
` declare under the penalty of perjury under the laws of the United States of America that
`the foregoing is true and correct.
`
`
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`
`
`_____________________________
`Signature
`
`August 9, 2018
`Date
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`
`
`Frank Reginald David Apps
`Name
`
`
`Mr.
`Title
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`
`
`
`
`
`
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`
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`MAIA Exhibit 1007
`MAIA V. BRACCO
`IPR PETITION
`
`

`

`{EER‘EEFECATE
`
`I, Flank Reginald David Apps, MITI, BA Hons Cantah., of Ingatestone
`Translations Ltd, 3 Crown Meadow, Braintree Essex CM7 '9EX, UK do
`hereby solemnly and sincerely declare as follows:
`1. I am .a hill—time translator of Japanese into English and am familiar
`with the English and Japanese languages;
`2. The following is a true translation, made by me, of PCT patent
`publication WOODS 1629.
`
`F.R.D. Apps
`25ill April 20-18
`
`
`
`
`

`

`1
`
`World Intellectual Property
`Organization
`International Office
`International Application
`published in accordance with
`Patent Cooperation Treaty
`A1
`
`
`
`
`
`
`
`PCT
`
`(51) International Patent
`Classification 7
`A61K 38/19, 38/22, 38/29,
`38/00, 47/16, 47/26, 47/14
`
`
`
`
`
`(11) International Publication
`WO00/51629
`(43) International Publication
`Date
`September 8, 2000 (08.09.00)
`
`
`(21) International Application Number
`(22) International Application Date
`(30) Priority Right Data
`Patent Application H 11/52314 March 1, 1999 (01.03.99)
`(71) Applicant (for all designated states except USA)
`Chugai Seiyaku Kabushiki Kaisha
`5-1, Ukima 5-chome, Kita-ku, Tokyo 115-8543 (JP)
`(72) Inventor; and
`(75) Inventor/applicant (for US only)
`SATO, Yasushi [JP/JP]
`c/o Chugai Seiyaku Kabushiki Kaisha, Tokyo (JP)
`41-8 Takada 3-chome, Toyoshima-ku, Tokyo 171-8545
` (74) Agent
`SHAMOTO, Ichio et al.
`Yuasa and Hara Patent Office, Section 206, New Ohtemachi Bldg., 2-1, Ohtemachi 2-chome,
`Chiyoda-ku, Tokyo 100-0004
`
`(81) Designated States AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE,
`DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU,
`LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA,
`UG, US, UZ, VN, YU, ZA, ZW. European Patent Office (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU,
`MC, NL, PT, SE) African Intellectual Property Organization (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR,
`NE, SN, TD, TG). African Regional Intellectual Property Organization (GH, GM, KE, LS, MW, SD, SL, SZ, TZ,
`UG, ZW) Eurasian Patent Organization (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM)
`
`Appended Published Document
`International Search Report
`
`(54) Title of the invention: LONG-TERM STABLE FORMULATION
`
`
`
`
`
`PCT/JP00/01160
`February 29, 2000 (29.02.00)
`
`JP
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`
`
`

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`2
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`DZ Algeria
`
`LC Saint Lucia
`
`EE Estonia
`ES Spain
`FI Finland
`FR France
`GA Gabon
`GB United Kingdom
`
`LI Lichtenstein
`LK Sri Lanka
`LR Liberia
`LS Lesotho
`LT Lithuania
`LU Luxembourg
`
`
`(57) Abstract
`A stable G-CSF formulation having a residual ratio of G-CSF of 90% or more after long-term
`storage testing at 25°C for 3 months or a residual ratio of G-CSF of 90% or more after long-term storage
`testing at 40°C for 2 months or a residual ratio of G-CSF of 90% or more after accelerated testing at 50°C
`for 1 month or a residual ratio of G-CSF of 90% or more after accelerated testing at 60°C for 2 weeks and
`a content of Met-oxidized G-CSF of 1% or less after accelerated testing at 50°C for 1 month or after
`accelerated testing at 60°C for 2 weeks. A method for suppressing the formation of the methionine-
`oxidized variant of a physiologically active protein having methionine residues characterized by adding
`methionine to a composition containing this protein is disclosed.
`
`
`Code used for identifying the PCT member countries listed on the first page of the
`international application pamphlet published under the PCT (reference information)
`AE United Arab
`DM Dominica
`KZ Kazakhstan
`
`emirates
`AG Antigua and
`Barbuda
`AL Albania
`AM Armenia
`AT Austria
`AU Australia
`AZ Azerbaijan
`BA Bosnia and
`Herzegovina
`BB Barbados
`BE Belgium
`BF Burkina Faso
`BG Bulgaria
`
`RU Russian
`Federation
`SD Sudan
`SE Sweden
`SG Singapore
`SI Slovenia
`SK Slovakia
`SL Sierra Leone
`
`SN Senegal
`SZ Swaziland
`TD Chad
`TG Togo
`
`TJ Tajikistan
`TM Turkmenistan
`
`TR Turkey
`TT Trinidad and
`Tobago
`TZ United Republic of
`Tanzania
`UA Ukraine
`UG Uganda
`US United States of
`America
`UZ Uzbekistan
`VN Vietnam
`
`GD Grenada
`GE Georgia
`GH Ghana
`GM Gambia
`
`BJ Benin
`BR Brazil
`
`BY Belarus
`CA Canada
`
`GN Guinea
`GR Greece
`
`GW Guinea-Bissau
`HR Croatia
`
`CF Central African
`Republic
`CG Congo
`CH Switzerland
`CI Côte d’Ivoire
`
`CM Cameroon
`CN China
`
`HU Hungary
`
`ID Indonesia
`IE Ireland
`IL Israel
`
`IN India
`IS Iceland
`
`LV Latvia
`MA Morocco
`MC Monaco
`MD Republic of
`Moldova
`MG Madagascar
`MK The former
`Yugoslav Republic of
`Macedonia
`ML Mali
`MN Mongolia
`
`MR Mauritania
`
`MW Malawi
`MX Mexico
`MZ Mozambique
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`NE Niger
`NL Netherlands
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`

`

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`CR Costa Rica
`CU Cuba
`CY Cyprus
`CZ Czechia
`DE Germany
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`DK Denmark
`
`
`3
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`IT Italy
`JP Japan
`KE Kenya
`KG Kyrgyzstan
`KP Democratic
`People’s Republic of
`Korea
`KR Republic of Korea
`
`
`
`NO Norway
`NZ New Zealand
`PL Poland
`PT Portugal
`RO Romania
`
`YU Yugoslavia
`ZA South Africa
`ZW Zimbabwe
`
`
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`
`
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`

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`4
`
`Specification
`Long-term stable formulation
`
`
`Technical field
`The present invention relates to a G-CSF (granulocyte colony-stimulating factor)
`formulation and in particular relates to a stable G-CSF preparation showing little loss of the
`active ingredient even after long-term storage, and wherein the content of Met-oxidized G-CSF
`is low.
`
`Background art
`G-CSF is a glycoprotein of molecular weight about 20,000, which acts on the precursor
`cells of neutrophils, promoting their proliferation and differentiation to maturation.
`The present applicant purified high-purity human G-CSF by culturing a cell line collected
`from tumor cells of a patient with cancer of the floor of the mouth. Prompted by this, the
`present applicant succeeded in cloning the human G-CSF gene. This has now made it possible to
`produce in large quantity recombinant human G-CSF by microorganisms or animal cells by
`genetic engineering techniques. Also, the present applicant succeeded in formulating this
`purified G-CSF and supplying this as a product to the market as an anti-infective agent
`(Japanese Patent Number 2116515).
`G-CSF is employed in extremely small quantity: usually a formulation containing G-CSF in
`the amount of 0.1~1000μg (preferably 5~500μg) is administered at a rate of 1~7 times/week,
`for an adult. However, this G-CSF tends to be adsorbed on the wall of the container, such as for
`example the injection ampoule or syringe. Also, G-CSF is unstable and susceptible to extrinsic
`factors such as temperature, humidity, oxygen, UV rays or the like to undergo physical or
`chemical changes including association, polymerization or oxidation, resulting in substantial loss
`of activity.
`Thus, various formulation designs were made to supply stable G-CSF formulations to the
`market. For example, formulations containing at least one member selected from the group
`consisting of (a) at least one amino acid selected from threonine, tryptophan, lysine,
`hydroxylysine, histidine, arginine, cysteine, cystine and methionine; (b) at least one sulfur-
`containing reducing agent; or (c) at least one antioxidant were proposed (Japanese Patent No.
`2577744). G-CSF formulations containing a surfactant such as a polysorbate as a stabilizer were
`also proposed (Laid-open Japanese Patent Application No. S 63-146826).
`Lyophilized G-CSF formulations containing maltose, raffinose, sucrose, trehalose or an
`aminosugar were also reported, which are advantageous from the viewpoint of limiting
`deposits on the container to suppress chemical changes (Japanese Translation of PCT
`Application No. H 8-504784).
`Some products currently on the market contain a protein commonly used as a stabilizer
`such as human serum albumin or purified gelatin for suppressing such chemical or physical
`changes. However, addition of a protein as a stabilizer had problems such as the necessity of a
`very complicated process for removing contamination with viruses.
`However, there is more production of Met-oxidized G-CSF in the absence of such a
`protein, leading to the problem of deterioration of product quality.
`Disclosure of the Invention
`
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`

`

`
`
`5
`
`An object of the present invention is to provide a G-CSF formulation, which is more
`stable even during long-term storage and which has a low content of Met-oxidized G-CSF.
`As a result of careful studies to achieve the above object, we accomplished the present
`invention on the basis of the finding that a G-CSF formulation showing a high residual ratio of
`G-CSF and a low content of Met-oxidized G-CSF even after long-term storage can be obtained
`by adding a combination of specific amino acids as a stabilizer.
`Specifically, the present invention provides a stable G-CSF formulation having a residual
`ratio of G-CSF of 90% or more after long-term storage testing at 25°C for 3 months or a residual
`ratio of G-CSF of 90% or more after long-term storage testing at 40°C for 2 months or a residual
`ratio of G-CSF of 90% or more after accelerated testing at 50°C for 1 month or a residual ratio of
`G-CSF of 90% or more after accelerated testing at 60°C for 2 weeks and a content of Met-
`oxidized G-CSF of 1% or less after accelerated testing at 50°C for 1 month or after accelerated
`testing at 60°C for 2 weeks.
`The present invention also provides said G-CSF formulation containing one or more
`amino acids selected from the group consisting of lysine, histidine, arginine, aspartic acid,
`glutamic acid, threonine and asparagine; one or more amino acids selected from hydrophobic
`amino acids; and methionine.
`The present invention also provides said G-CSF formulation wherein said hydrophobic
`amino acid is selected from phenylalanine, tryptophan and leucine.
`The present invention also provides said G-CSF formulation containing one or more
`amino acids selected from the group consisting of lysine, histidine, arginine, aspartic acid and
`glutamic acid; one or more amino acids selected from the group consisting of phenylalanine,
`tryptophan and leucine; and methionine.
`The present invention also provides said G-CSF formulation containing phenylalanine,
`arginine and methionine.
`The present invention also provides said G-CSF formulation substantially free from
`protein as a stabilizer.
`The present invention also provides said G-CSF formulation in the form of a lyophilized
`formulation.
`The present invention also provides said G-CSF formulation further containing mannitol.
`The present invention also provides said G-CSF formulation further containing a
`surfactant.
`The present invention also provides said G-CSF formulation wherein said surfactant is a
`polyoxyethylene sorbitan alkyl ester.
`The present invention also provides said G-CSF formulation wherein said surfactant is
`polysorbate 20 and/or 80.
`The present invention also provides said G-CSF formulation having a pH of 5~7.
`The present invention also provides said G-CSF formulation having a pH of 5.5~6.8.
`The present invention also provides said G-CSF formulation having a pH of 6.5.
`The present invention also provides said G-CSF formulation wherein G-CSF is produced
`from CHO cells.
`The present invention also provides a stable G-CSF formulation having a residual ratio of
`G-CSF of 90% or more after long-term storage testing at 25°C for 3 months or a residual ratio of
`G-CSF of 90% or more after long-term storage testing at 40°C for 2 months or a residual ratio of
`
`
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`

`

`6
`
`
`G-CSF of 90% or more after accelerated testing at 50°C for 1 month or a residual ratio of G-CSF
`of 90% or more after accelerated testing at 60°C for 2 weeks, characterized in that it contains
`one or more amino acids selected from the group consisting of lysine, histidine, arginine,
`aspartic acid, glutamic acid, threonine and asparagine; and one or more amino acids selected
`from hydrophobic amino acids; and it has a pH of 5~7.
`The present invention also provides a stable G-CSF formulation having a residual ratio of
`G-CSF of 90% or more after long-term storage testing at 25°C for 3 months or a residual ratio of
`G-CSF of 90% or more after long-term storage testing at 40°C for 2 months or a residual ratio of
`G-CSF of 90% or more after accelerated testing at 50°C for 1 month or a residual ratio of G-CSF
`of 90% or more after accelerated testing at 60°C for 2 weeks, characterized in that it contains
`one or more amino acids selected from the group consisting of lysine, histidine, arginine,
`aspartic acid and glutamic acid; and one or more amino acids selected from the group
`consisting of phenylalanine, tryptophan and leucine; and it has a pH of 5~7.
`The present invention also provides either one of said G-CSF formulations having a pH of
`
`6.5.
`
`The present invention also provides a method for inhibiting a physiologically active
`protein containing a methionine residue from producing a variant oxidized at the methionine
`residue, comprising adding methionine to a composition containing said protein.
`The present invention also provides said method wherein said physiologically active
`protein is a cytokine or a physiologically active peptide.
`The present invention also provides said method wherein said physiologically active
`protein is a colony-stimulating factor or PTH.
`The present invention also provides said method wherein said physiologically active
`protein is G-CSF, erythropoietin or PTH.
`The present invention also provides said method wherein other proteins are not present
`as stabilizers.
`The present invention also provides said method wherein said composition containing a
`physiologically active protein having a methionine residue is lyophilized or in the form of a
`solution.
`The present invention also provides a stabilized composition containing a physiologically
`active protein having a methionine residue, further containing methionine and one or more
`other amino acids.
`The present invention also provides said stabilized composition containing a
`physiologically active protein having a methionine residue wherein the amino acid is one or
`more selected from the group of consisting of lysine, histidine, arginine, aspartic acid, glutamic
`acid, phenylalanine, tryptophan, leucine, isoleucine, valine, alanine, proline, glycine, serine,
`threonine, asparagine, glutamine and tyrosine.
`The present invention also provides said stabilized composition containing a
`physiologically active protein having a methionine residue, characterized in that it is free from
`other proteins as stabilizers.
`Brief Description of the Drawings
`FIG. 1 shows chromatograms of samples 34 and 36 as assayed by method 2 described
`later after accelerated testing at 60°C for 2 weeks.
`
`
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`

`
`
`7
`
`FIG. 2 shows chromatograms of samples 34~36 as assayed by method 2 described later
`immediately after preparation and after accelerated testing at 50°C for 1 month.
`FIG. 3 shows HPLC chromatograms of parathyroid hormone solution formulations as
`assayed by the method shown in example 6 (storage at 50°C for 3 days), which demonstrate
`that addition of methionine has an inhibitory effect against oxidation of methionine residues. In
`FIG. 3, the highest peaks at the center correspond to intact PTH, and peaks designated as Met-8
`and Met-18 correspond to PTH oxidized at the 8th methionine residue and the 18th methionine
`residue, respectively.
`The Most Preferred Embodiments of the Invention
`G-CSF used for formulations of the present invention includes any high-purity human G-
`CSF. Specifically, it may be derived from natural sources or obtained by genetic recombination
`so long as it has substantially the same biological activity as that of mammalian, particularly
`human G-CSF. Genetically recombinant G-CSF may have the same amino acid sequence as that
`of natural G-CSF or may contain deletion, substitution or addition of one or more amino acids in
`said amino acid sequence so long as it has said biological activity. G-CSF in the present invention
`may be prepared by any process, e.g., it may be extracted and purified by various techniques
`from cultures of a human tumor cell line or may be produced by genetic engineering in cells of
`E. coli, yeast; Chinese hamster ovary (CHO), C127 or the like cells of animal origin and then
`extracted and purified by various techniques. Preferably G-CSF is produced in E. coli, yeast or
`CHO cells by genetic recombination. Most preferably, G-CSF is produced in CHO cells by genetic
`recombination.
`Preferably, G-CSF formulations of the present invention are substantially free from
`protein such as human serum albumin or purified gelatin as a stabilizer.
`G-CSF formulations of the present invention are very stable as compared with previously
`known G-CSF formulations as they have a residual ratio of G-CSF of 90% or more, preferably
`95% or more after long-term storage testing at 25°C for 3 months, or a residual ratio of G-CSF of
`90% or more, preferably 95% or more after long-term storage testing at 40°C for 2 months, or a
`residual ratio of G-CSF of 90% or more, preferably 95% or more after accelerated testing at
`50°C for 1 month, or a residual ratio of G-CSF of 90% or more, preferably 95% or more after
`accelerated testing at 60°C for 2 weeks and a content of Met-oxidized G-CSF of 1% or less,
`preferably below the detection limit, after accelerated testing at 50°C for 1 month or after
`accelerated testing at 60°C for 2 weeks.
`An example of G-CSF formulations of the present invention is a G-CSF formulation
`containing one or more amino acids selected from the group consisting of lysine, histidine,
`arginine, aspartic acid, glutamic acid, threonine and asparagine, preferably one or more amino
`acids selected from the group consisting of lysine, histidine, arginine, aspartic acid and glutamic
`acid; one or more amino acids selected from hydrophobic amino acids, preferably one or more
`amino acids selected from the group consisting of phenylalanine, tryptophan and leucine; and
`methionine.
`Another example of G-CSF formulations of the present invention is a stable G-CSF
`formulation having a residual ratio of G-CSF of 90% or more after long-term storage testing at
`25°C for 3 months or a residual ratio of G-CSF of 90% or more after long-term storage testing at
`40°C for 2 months or a residual ratio of G-CSF of 90% or more after accelerated testing at 50°C
`for 1 month or a residual ratio of G-CSF of 90% or more after accelerated testing at 60°C for 2
`
`
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`

`8
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`
`weeks and a content of Met-oxidized G-CSF of 1% or less after accelerated testing at 50°C for 1
`month or after accelerated testing at 60°C for 2 weeks, characterized in that it contains one or
`more amino acids selected from the group consisting of lysine, histidine, arginine, aspartic acid,
`glutamic acid, threonine and asparagine, preferably one or more amino acids selected from the
`group consisting of lysine, histidine, arginine, aspartic acid and glutamic acid; one or more
`amino acids selected from hydrophobic amino acids, preferably one or more amino acids
`selected from the group consisting of phenylalanine, tryptophan and leucine; and methionine;
`and it has a pH of 5~7.
`Amino acids used in the present invention include free amino acids and salts thereof
`such as sodium salts, potassium salts and hydrochlorides. Formulations of the present invention
`may contain D-, L- and DL-variants of these amino acids, more preferably L-variants and salts
`thereof.
`The amount of amino acids to be added to formulations of the present invention can be
`determined in a preferred range using the test method described later depending on the type
`of the amino acid used. Generally, a final dose of 0.001~50 mg/ml is added. For example,
`phenylalanine is preferably added at 0.1~25 mg/ml, more preferably 1~20 mg/ml, arginine is
`preferably added at 0.1~25 mg/ml, more preferably 1~20 mg/ml, and methionine is preferably
`added at 0.001~5 mg/ml, more preferably 0.01~4 mg/ml.
`Formulations of the present invention may contain isotonizing agents, e.g., polyethylene
`glycol; and sugars such as dextran, mannitol, sorbitol, inositol, glucose, fructose, lactose, xylose,
`mannose, maltose, sucrose and raffinose. Mannitol is especially preferred. The amount of
`mannitol added into formulations is 1~100 mg/ml, more preferably 5~60 mg/ml.
`Formulations of the present invention may further contain surfactants. Typical examples
`of surfactants include: nonionic surfactants, e.g., sorbitan fatty acid esters such as sorbitan
`monocaprylate, sorbitan monolaurate, sorbitan monopalmitate; glycerin fatty acid esters such
`as glycerin monocaprylate, glycerin monomyristate, glycerin monostearate; polyglycerin fatty
`acid esters such as decaglyceryl monostearate, decaglyceryl distearate, decaglyceryl
`monolinoleate; polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene sorbitan
`monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate,
`polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene
`sorbitan tristearate; polyoxyethylene sorbitol fatty acid esters such as polyoxyethylene sorbitol
`tetrastearate, polyoxyethylene sorbitol tetraoleate; polyoxyethylene glycerin fatty acid esters
`such as polyoxyethylene glyceryl monostearate; polyethylene glycol fatty acid esters such as
`polyethylene glycol distearate; polyoxyethylene alkyl ethers such as polyoxyethylene lauryl
`ether; polyoxyethylene polyoxypropylene alkyl ethers such as polyoxyethylene
`polyoxypropylene glycol ether, polyoxyethylene polyoxypropylene propyl ether,
`polyoxyethylene polyoxypropylene cetyl ether; polyoxyethylene alkyl phenyl ethers such as
`polyoxyethylene nonyl phenyl ether; polyoxyethylene hardened castor oils such as
`polyoxyethylene castor oil, polyoxyethylene hardened castor oil (polyoxyethylene
`hydrogenated castor oil); polyoxyethylene beeswax derivatives such as polyoxyethylene
`sorbitol beeswax; polyoxyethylene lanolin derivatives such as polyoxyethylene lanolin;
`polyoxyethylene fatty acid amides such as polyoxyethylene stearic acid amide having an HLB of
`6~18; anionic surfactants, e.g., alkyl sulfates having a C10~18 alkyl group such as sodium
`cetylsulfate, sodium laurylsulfate, sodium oleylsulfate; polyoxyethylene alkyl ether sulfates
`
`
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`

`

`9
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`
`having an average EO mole number of 2~4 and a C10~18 alkyl group such as sodium
`polyoxyethylene laurylsulfate; alkyl sulfosuccinic acid ester salts having a C8~18 alkyl group
`such as sodium laurylsulfosuccinate; and natural surfactants, e.g., lecithin;
`glycerophospholipids; sphingophospholipids such as sphingomyelin; and sucrose fatty acid
`esters of C12~18 fatty acids. One or two or more of these surfactants may be added in
`combination to formulations of the present invention.
`Preferred surfactants are polyoxyethylene sorbitan fatty acid esters, more preferably
`polysorbates 20, 21, 40, 60, 65, 80, 81, 85, most preferably polysorbates 20 and 80.
`The amount of surfactants to be added to G-CSF-containing formulations of the present
`invention is typically 0.0001-10 parts by weight per part by weight of G-CSF, preferably 0.01~5
`parts by weight per part by weight of G-CSF, and most preferably 0.2~2 parts by weight per part
`by weight of G-CSF.
`Preferably, G-CSF formulations of the present invention have a pH of 5~7, more
`preferably 5.5~6.8, still more preferably 6~6.7, and most preferably 6.5.
`G-CSF formulations of the present invention may further contain diluents, solubilizing
`agents, excipients, pH-modifiers, soothing agents, buffers, sulfur-containing reducing agents,
`antioxidants or the like, if desired. For example, sulfur-containing reducing agents include N-
`acetylcysteine, N-acetylhomocysteine, thioctic acid, thiodiglycol, thioethanolamine,
`thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate, glutathione,
`and sulfhydryl-containing compounds such as thioalkanoic acids having 1~7 carbon atoms.
`Antioxidants include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-
`tocopherol, tocopherol acetate, L-ascorbic acid and salts thereof, L-ascorbyl palmitate, L-
`ascorbyl stearate, sodium bisulfite, sodium sulfite, triamyl gallate, propyl gallate or chelating
`agents such as disodium ethylenediamine tetraacetate (EDTA), sodium pyrophosphate, or
`sodium metaphosphate. Other components commonly added may also be contained, e.g.,
`inorganic salts such as sodium chloride, potassium chloride, calcium chloride, sodium
`phosphate, potassium phosphate, sodium bicarbonate; and organic salts such as sodium citrate,
`potassium citrate, or sodium acetate.
`G-CSF formulations of the present invention include solution formulations, lyophilized
`formulations, spray-dried formulations, etc. Lyophilized formulations are most preferred.
`Formulations of the present invention can be prepared by dissolving these components
`in an aqueous buffer known in the art of solution formulations such as phosphate buffers
`(preferably sodium monohydrogen phosphate-sodium dihydrogen phosphate system) and/or
`citrate buffers (preferably sodium citrate buffer) to prepare a solution formulation, or
`lyophilizing or spray-drying a thus prepared solution formulation by standard procedures.
`Stabilized G-CSF-containing formulations of the present invention are normally
`administered via parenteral routes such as injection (subcutaneous, intravenous or
`intramuscular injection) or percutaneous, mucosal, nasal or pulmonary administration, but may
`also be orally administered.
`G-CSF formulations of the present invention are normally packed in a sealed and
`sterilized plastic or glass container, and dissolved in pure water (sterilized water for injection)
`before use.
`The amount of G-CSF contained in formulations of the present invention can be
`determined depending on the type of the disease to be treated, the severity of the disease, the
`
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`

`10
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`
`age of the patient or other factors, but generally ranges from 1 to 1000 μg/mL, preferably 10 to
`800 μg/mL, more preferably 50 to 500 μg/mL.
`Formulations of the present invention are clinically very useful as they were found to
`improve protective functions based on immune response, such as patient resistance or activity,
`when they were coadministered with drugs such as antibiotics, antibacterial agents or
`anticancer agents in the chemotherapy of infectious diseases or cancer. Therefore,
`formulations of the present invention can be administered in combination with these drugs.
`As demonstrated in the examples below, G-CSF formulations of the present invention
`show a very good residual ratio of G-CSF after long-term storage testing at 25°C for 3 months or
`long-term storage testing at 40°C for 2 months or accelerated testing at 50°C for 1 month or
`accelerated testing at 60°C for 2 weeks. Moreover, scarcely any content of Met-oxidized G-CSF
`was observed after accelerated testing at 50°C for 1 month or accelerated testing at 60°C for 2
`weeks. G-CSF formulations of the present invention have a residual ratio of C-CSF of 90% or
`more, preferably 95% or more after long-term storage testing at 25°C for 3 months, or a
`residual ratio of G-CSF of 90% or more, preferably 95% or more after long-term storage testing
`at 40°C for 2 months, or a residual ratio of G-CSF of 90% or more, preferably 95% or more after
`accelerated testing at 50°C for 1 month, or a residual ratio of G-CSF of 90% or more, preferably
`95% or more after accelerated testing at 60°C for 2 weeks and a content of Met-oxidized G-CSF
`of 1% or less, preferably below the detection limit, after accelerated testing at 50°C for 1 month
`or after accelerated testing at 60°C for 2 weeks.
`It was observed from the results of the examples below that the residual ratio of G-CSF
`in formulations of the present invention after long-term storage in particular at normal
`temperatures can be improved by adding one or more amino acids selected from the group
`consisting of lysine, histidine, arginine, aspartic acid, glutamic acid, threonine and asparagine
`and one or more amino acids selected from hydrophobic amino acids and that the content of
`Met-oxidized G-CSF can be kept below the detection limit by adding methionine. Without
`wishing to be bound to any specific theory, we assumed that the added methionine is oxidized
`in place of methionine residues of G-CSF so that the content of Met-oxidized G-CSF decreases.
`According to the present invention, when methionine is added to a composition of a
`physiologically active protein having a methionine residue, which has a physiological activity
`even in a small amount, but is comparatively susceptible to production of a variant oxidized at
`the methionine residue, said physiologically active protein can be prevented from producing
`the variant oxidized at the methionine residue. Addition of methionine seems to be especially
`effective when said physiologically active protein composition is free from other proteins as
`stabilizers or when said protein composition is lyophilized or in the form of a solution because
`the variant of said protein oxidized at the methionine residue is more likely to be produced in
`such cases.
`When one or more other amino acids are added to a composition of the present
`invention, a stabilized composition containing a physiologically active protein having a
`methionine residue can also be prepared, which is inhibited from producing a variant oxidized
`at the methionine residue as well as inhibited from decomposition, aggregation or the like of
`said physiologically active protein.
`Amino acids that can be added for this purpose include lysine, histidine, arginine,
`aspartic acid, glutamic acid, phenylalanine, tryptophan, leucine, isoleucine, valine, alanine,
`
`
`
`
`

`

`11
`
`
`
`Phenylalanine Arginine Methionine Mannitol
`
`
`proline, glycine, serine, threonine, asparagine, glutamine and tyrosine, preferably histidine,
`arginine and phenylalanine.
`Physiologically active proteins of the present invention include, for example:
`cytokines such as interleukins (e.g., IL-1 ~ IL-13), colony-stimulating factors (e.g.,
`granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF),
`granulocyte/macrophage colony-stimulating factor (GM-CSF), erythropoietin (EPO)), interferons
`(e.g., IFN-α, β, γ), tumor necrosis factors (e.g., TNF-α, TNF-β), transforming growth factor (TGF),
`platelet-derived growth factor (PDGF), LIF (leukemia inhibitory factor), oncostatin M (OSM),
`migration inhibitory factor (MIF), chemokines (e.g., IL-8, LD78, MCP-1); physiologically active
`peptides such as insulin, glucagon, parathyroid hormone (PTH), gastrin, selectin,
`cholecystokinin, gastric inhibitory polypeptides, substance P, motilin, spleen polypeptides,
`neurotensin, enteroglucagon, gastrin-releasing peptides, somatostatin-28, dynorphin, galanin,
`balenin, pancreostatin and zeopsin; bioenzymes such as enzymes having a methionine residue
`at the active center (e.g., malate dehydrogenase); or variants thereof.
`Physiologically active proteins of the present invention are preferably cytokines or
`physiologically active peptides, more preferably colony-stimulating factors such as G-CSF or
`erythropoietin or PTH, still more preferably G-CSF, erythropoietin or PTH.
`The following examples further illustrate the present invention, without limiting the
`same thereto. Various changes and modifications can be made by those skilled in the art on the
`basis of the description of the invention, and such changes and modifications are also included
`in the present invention.
`Examples
`Test Method
`Formulated solutions containing various components in the amounts shown in Tables 1
`and 2 below were prepared and aseptic-filtered, and then precisely 1 mL each was aseptically
`packed in a vial and lyophilized. After completion of lyophilization, the vial was completely
`capped to prepare G-CSF lyophilized formulations.
`
`[Table 1]
`Table 1
`G-CSF
`
`10 mg
`
`10 mg
`
`0
`
`50 mg
`
`10 mg
`
`10 mg
`
`0
`
`50 mg
`
`0.1 mg
`
`Sample
`1
`
`250
`μg
`
`Sample
`2
`
`250
`μg
`
`
`
`
`
`Polysorbate
`20
`0.1 mg
`
`pH b