O
`
`United States Patent (19)
`Takruri
`
`USOO527235A
`
`(11) Patent Number:
`(45) Date of Patent:
`
`5,272,135
`Dec. 21, 1993
`
`54 METHOD FOR THE STABILIZATION OF
`OTHER PUBLICATIONS
`METHONINE-CONTAINING
`CA 100:144972z, Green Cross.
`POLYPEPTOES
`J. Parenteral Science and Technology, 42:1988, “Paren
`h,
`akruri, N
`teral Formulations of Peptides, Proteins and Stabiliz
`75) Inventor E" Takruri, Newport Beac
`ers", Chay et al., S3-S26.
`73 Assignee: Chiron Ophthalmics, Inc., Irvine,
`Primary Examiner-Jeffrey E. Russel
`Calif.
`Assistant Examiner-P. Lynn Touzeau
`Attorney, Agent, or Firm-Rothwell, Figg, Ernst & Kurz
`21) Appl. No.: 663,021
`(57)
`ABSTRACT
`22 Filed:
`Mar. 1, 1991
`51) int. Cl. ....................... C07K 15/00; C07K 17/00 A method for inhibiting the oxidation of a polypeptide
`52) U.S. Cl. ........................................ 514/12; 514/21;
`in a liquid or semi-solid pharmaceutical or therapeutic
`530/399
`preparation, the polypeptides having an amino acid
`(58) Field of Search ............... 530/397, 399, 300, 350,
`sequence comprising at least one methionine residue,
`530/345; 514/12, 21, 2, 8,973
`wherein the amino acid methionine is added in a suffi
`(56)
`References Cited
`cient amount to inhibit the oxidation of the methionine
`residue(s) to methionine sulfoxide.
`U.S. PATENT DOCUMENTS
`4,835,254 5/1989 DiMarchi et al. .................. 530/345
`
`25 Claims, 4 Drawing Sheets
`
`
`
`FORN
`FORM II
`FORM II
`FORN M
`
`zszk
`
`2
`
`ZN 3. WN WN N %
`
`MAIA Exhibit 1032
`MAIA V. BRACCO
`IPR PETITION
`
`

`

`U.S. Patent
`
`Dec. 21, 1993
`
`Sheet 1 of 4
`
`5,272,135
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`
`
`
`
`
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`
`PERCENT OXIDATION
`
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`
`

`

`U.S. Patent
`
`Dec. 21, 1993
`
`Sheet 2 of 4
`
`5,272,135
`
`
`
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`g
`2
`ZZZZZZZZZZ
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`t
`
`PERCENT OXDATION
`
`

`

`U.S. Patent
`
`Dec. 21, 1993
`
`Sheet 3 of 4
`
`5,272,135
`
`nAU
`
`300
`
`2OO
`
`00
`
`5
`
`O
`
`5
`TIME (MIN.)
`
`2O
`
`25
`
`FIG. 3A
`
`D
`
`mAU
`
`160
`
`40
`20
`
`f00
`80
`
`60
`40
`20
`
`5
`
`O
`
`5
`TIME (MIN)
`
`2O
`
`25
`
`FIG. 3B
`
`

`

`U.S. Patent
`
`Dec. 21, 1993
`
`Sheet 4 of 4
`
`5,272,135
`
`OO
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`80
`
`60
`
`40
`
`2O
`
`O
`
`FIG. 4A
`
`
`
`5
`
`O
`
`5
`TIME (MIN)
`
`20
`
`25
`
`5
`
`O
`
`15
`TIME (MN)
`
`20
`
`25
`
`

`

`1.
`
`METHOD FOR THE STABILIZATION OF
`METHONINE-CONTAINING POLYPEPTIDES
`
`5
`
`10
`
`15
`
`50
`
`BACKGROUND OF THE INVENTION
`This invention relates to a method of inhibiting the
`oxidation of a polypeptide in a liquid or semi-solid me
`dium, the polypeptide having an amino acid sequence
`comprising at least one methionine residue. More spe
`cifically, the invention relates to a method of inhibiting
`the oxidation of a tissue growth factor in a liquid or
`semi-solid medium, wherein the amino acid sequence of
`the tissue growth factor comprises at least one methio
`nine residue. The invention further relates to stabilized,
`pharmaceutically effective preparations of such poly
`peptides.
`In recent years, researchers have developed numer
`ous techniques that have made possible the production
`and purification of various polypeptides on a commer
`cial scale for therapeutic and pharmaceutical purposes.
`20
`For example, polypeptides such as epidermal growth
`factor can be employed as the pharmacologically active
`component in ophthalmic preparations employed to
`enhance the repair of ocular tissue and also in cornea
`storage media employed in preserving eye tissue prior
`25
`to surgical transplantation, i.e., keratoplasty. In particu
`lar, epidermal growth factor has been shown to have
`wound healing promoting activity (Plast. Reconstr.
`Surg., 64,766 (1979); J. Surg. Res., 33,164 (1982)); anti
`inflammatory activity and analgesic activity (Japanese
`30
`Laid-open Patent Publication No. 115785/1985). Epi
`dermal growth factor is just one of a number of growth
`factors which are low molecular weight polypeptides
`that have the ability to stimulate the repair and matura
`tion of tissue when applied thereto.
`35
`A growth factor can be selective with regard to both
`the type of tissue it acts upon as well as the extent of
`stimulation it causes in responsive cell types. For exam
`ple, epidermal growth factor (EGF), vaccinia growth
`factor (VGF), nerve growth factor (NGF), platelet
`derived growth factor (PDGF), fibroblast growth fac
`tor (FGF), transforming growth factor alpha (TGF-a),
`transforming growth factor beta (TGF-6), and insulin
`like growth factor I (IGF-I) are all low molecular
`weight polypeptides which possess the ability to stimu
`45
`late the division and naturation of certain cells.
`The growth factors are known proteins, the proper
`ties and biological activities of which have been are
`described, for example, in review articles by Krisis et
`al., Biotechnology, February, 1985, pp. 135-140 and in
`Hormonal Proteins and Peptides, Ed. by Chao Hao Li,
`Vol 12, "Growth Factors' Academic Press (1984).
`Epidermal growth factor is a low molecular weight
`protein (6040 daltons has been reported) previously
`isolated from mouse salivary glands according to the
`method of Savage and Cohen, J. Biol. Chem., 1972; 257;
`7609-11. European Patent Office publication number
`EP 177,915 teaches the production of recombinant
`human EGF (rhEGF) by E. coli transformed with a
`vector containing DNA encoding EGF,
`60
`A process for obtaining transforming growth factor
`alpha is described in J.E. DeLarco and G.E. Todaro,
`"Growth Factors From Marine Carcoma Virus-Trans
`formed Cells', Proc.
`Natl. Acad. Sci.
`U.S.A.,
`75:4001-4005, 1978.
`EP 219,814 teaches the production of recombinant
`human insulin-like growth factor. I.E. Rinderknecht
`and R.E. Humbel teach the production of insulin-like
`
`5,272,135
`2
`growth factor I in "Polypeptides with the Non-Sup
`pressible Insulin-Like and Cell-Growth Promoting Ac
`tivities in Human Serum: Isolation, Chemical Charac
`terization and Some Biological Properties of Forms I
`and II", Proc. Natl. Acad. Sci. U.S.A., 73:2365-2369,
`1976.
`Vaccinia growth factor is obtained from vaccinia
`virus-infected cells according to the methods of D.R.
`Twardzik, J.P. Braun, J.E. Ranchalis, G.E. Todaro and
`B. Moss, "Vaccinia Virus-Infected Cells Release A
`Novel Polypeptide Functionally Related To Trans
`forming and Epidermal Growth Factors', Proc. Natl.
`Acad. Sci. U.S.A., 82:5300-5304, 1985.
`Pharmaceutical preparations containing growth fac
`tors in an aqueous medium, such as ophthalmic prepara
`tions of EGF, commonly are packaged in plastic con
`tainers made of low density polyethylene (LDPE) or
`polypropylene for convenient storage and application.
`However, these plastic containers are readily permeable
`to oxygen. The oxygen causes rapid oxidation of the
`methionine residue(s) in the growth factor to methio
`nine sulfoxide. It is the side chain of the methionine
`residue which is particularly vulnerable to oxidation
`(Manning et al., Pharmaceutical Research, Vol. 6, No.
`11, 1989). Although the growth factor is still biologi
`cally active after its methionine residues have been
`oxidized to methionine sulfoxide, the growth factor is
`not acceptable for pharmaceutical use according to the
`standards of regulatory agencies, such as the FDA,
`when high levels of methionine sulfoxide are present.
`Current precautionary procedures to try to exclude
`oxygen during the manufacture and packaging of the
`preparation have proven to be ineffective in preventing
`significant methionine oxidation. The result is that the
`pharmaceutical preparation has a shorter effective life
`than is potentially possible if the oxidation reaction
`could be inhibited. In addition to growth factors, oxida
`tion has been observed in many methionine containing
`peptide hormones during their isolation, synthesis and
`storage. Examples of some of the polypeptide hormones
`include adrenocorticotropic hormone, human growth
`hormone or somatotropin and the like.
`Certain amino acids and various combinations thereof
`and surfactants, such as polysorbate and poloxanner and
`the like have been used to stabilize peptide and protein
`compositions. See, for example, Yu-Chang, John Wang
`and Musetta A. Hansen, "Parenteral Formulations of
`Proteins and Peptides: Stability and Stabilizers', Jour
`nal of Parenteral Science and Technology, 42:S14, 1988.
`However, none of the amino acids or surfactants are
`used to deter the oxidation of methionine residues to
`methionine sulfoxide in a liquid or semi-solid medium.
`Therefore, there is a need for a method of inhibiting
`the oxidation in pharmaceutical vehicles of polypep
`tides having an amino acid sequence which comprises at
`least one methionine residue.
`SUMMARY OF THE INVENTION
`In accordance with the present invention, a method
`for inhibiting the oxidation of a polypeptide character
`ized by an amino acid sequence comprising at least one
`methionine residue comprises adding methionine to the
`medium in an amount effective to inhibit the oxidation
`of the methionine residue. The added methionine stabi
`lizes the preparation containing the polypeptide by
`inhibiting the rapid oxidation of the methionine resi
`due(s) to methionine sulfoxide. Polypeptides that can be
`
`55
`
`65
`
`

`

`10
`
`15
`
`5,272,135
`3
`4.
`stabilized in this manner include the tissue growth fac
`antibiotic (neomycin sulfate) and a synthetic steroid
`tors. In the preferred embodiment, the polypeptide is an
`(dexamethasone phosphate) accelerate the rate of cor
`epidermal growth factor.
`neal epithelial regeneration and significantly increase
`the strength of full-thickness stromal incisions in pri
`BRIEF DESCRIPTION OF THE DRAWINGS
`mates. P. Woost et al., "Acceleration of Corneal
`FIG. 1 illustrates the oxidation rates of four different
`Wound Healing', Proc. Int, Soc, for Eve Research III,
`formulations of recombinant human epidermal growth
`1984.
`factor (rhEGF) acetate at 4 C. over a 105 day time
`The amount of epidermal growth factor present in
`period.
`such ophthalmic preparations ranges from about
`FIG, 2 illustrates the oxidation rates of four different
`0.001% w/v to about 0.10% w/v of the composition.
`formulations of recombinant human epidermal growth
`The epidermal growth factor employed can be natu
`factor (rhEGF) acetate at room temperature (20' C.)
`rally occurring, such as bovine, rat, porcine, mouse or
`over a 105 day time period.
`human growth factor and the like, or it can be biosyn
`FIGS. 3A and 3B illustrate the high performance
`thetic, such as recombinant human epidermal growth
`liquid chromatogram (HPLC) for a formulation of
`factor (rhEGF).
`rhEGF and methionine at 4 C. on day 0 and on day
`Other growth factors which can be employed in
`105, respectively.
`pharmaceutical and therapeutic compositions and stabi
`FIGS. 4A and 4B illustrate the high performance
`lized by the method of this invention include methio
`liquid chromatogram (HPLC) for a formulation of
`nine containing insulin-like growth factor I (IGF-I),
`rhEGF without methionine at 4 C. on day 0 and on day
`transforming growth factor alpha precursor (TGF-aP),
`20
`105.
`transforming growth factor beta (TGF-3), transform
`ing growth factor beta precursor (TGF-gP), nerve
`growth factor (NGF), platelet-derived growth factor
`(PDGF), fibroblast growth factor (FGF), and vaccinia
`growth factor (VGF) as well as any methionine-con
`taining fragments, precursors and analogs thereof. The
`quantities employed vary and are known by those
`skilled in the art.
`The methionine is added to these compositions in
`amounts sufficient to significantly inhibit oxidation of
`the methionine residue(s) such that the amount of me
`thionine sulfoxide is acceptable to regulatory agencies.
`Typically, this means that the composition contains no
`more than about 10% to about 30% methionine sulfox
`ide. Generally, this can be achieved by adding methio
`nine such that the ratio of methionine added to methio
`nine residues ranges from about 10:1 to about 100:1.
`Accordingly, methionine is added to the composition in
`amounts such that its concentration ranges from about
`0.01% w/v to about 1.0% w/v. Preferably, the final
`concentration of the added methionine ranges from
`about 0.01% w/v to about 0.3% w/v.
`Adjuvants or pharmaceutically inactive ingredients
`can be added to the ophthalmic composition to further
`stabilize, preserve and maintain the composition. The
`adjuvants added to the composition are those which are
`typically added to aqueous ophthalmic preparations as
`practiced according to the art. For example, sodium
`chloride can be added at a concentration of about 0.9%
`w/v or less to assist in making the ophthalmic composi
`tion physiologically isotonic. Other components that
`optionally can be included in the composition include a
`buffer system composed of phosphates, citrates, ace
`tates, lactates and the like as practiced in the art to
`maintain a pH of optimum stability. Sodium hydroxide,
`hydrochloric acid or acetic acid and the like can be
`added to adjust the composition to the desired pH and
`viscosity agents, such as sodium carboxymethyl cellu
`lose, hydroxypropylmethyl cellulose or polyvinyl alco
`hol, at a concentration of about 2.0% w/v or less, and
`the like also can be added. Emulsifying agents such as
`povidone, at a concentration of about 5.0% w/v or less,
`and the like optionally can be added. Glycerin at a
`concentration of about 2.0% w/v to about 4% w/v can
`be added as a tonicity agent in situations where sodium
`chloride can not be used. Polymeric quaternary ammo
`nium salts at a concentration of about 0.005% w/v to
`about 0.001% w/v can be added as preservatives. Other
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`In accordance with the present invention, the addi
`25
`tion of the amino acid methionine to liquid or semi-solid
`compositions comprising methionine-containing poly
`peptides stabilizes the compositions. Such compositions
`include, for example, therapeutic and pharmaceutical
`preparations and tissue storage media. The added methi
`30
`onine stabilizes the preparation by inhibiting the oxida
`tion of the polypeptide's methionine residue(s) to methi
`online sulfoxide.
`As used herein, the term "polypeptide' encompasses
`natural, synthetic and recombinant polypeptides having
`35
`a desired biological activity, including polypeptides
`having deleted, replaced or altered amino acid sequen
`ces in comparison with the full-length natural polypep
`tide or biologically active fragments thereof,
`In practicing the preferred embodiment of this inven
`40
`tion, methionine is added to a pharmaceutical prepara
`tion, such as an aqueous ophthalmic preparation, com
`prising a methionine-containing growth factor, such as
`epidermal growth factor, in sufficient quantity to inhibit
`the oxidation of the methionine residue to methionine
`45
`sulfoxide. Any stereoisomer of methionine (L, D or DL
`isomer) or combinations thereof can be used. The result
`ing ophthalmic composition remains stable for a longer
`period of time than if methionine had not been added.
`The method and composition of the present invention
`50
`will be further described with particular focus on oph
`thalmic aqueous-based compositions of a growth factor
`such as EGF. It is to be understood, however, that the
`present invention can be used by those with ordinary
`skill in the art to stabilize other aqueous or semi-solid
`polypeptide compositions.
`Ophthalmic compositions comprising EGF can be
`used for the restorative process of the corneal epithe
`lium in nondystrophic diseases of the eye, for example
`herpetic lesions. See, for example, Salvatore Daniele et
`al., “The Effect of the Epidermal Growth Factor
`(EGF) on the Corneal Epithelium in Humans", Albrecht
`v. Graefer Arch. Klin. exp. Ophthalmologie, 210:159-165
`(1979). In addition, ophthalmic preparations can be
`used in combination with corticosteroids to accelerate
`65
`epithelial regeneration or healing of stromal wounds.
`Further, topical administration of biosynthetic human
`epidermal growth factor given in combination with an
`
`55
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`

`5,272,135
`S
`6
`preservatives that are characteristically employed as
`nine ranges from about 0.001% w/v to about 0.003%
`such for ophthalmic preparations also can be included
`w/v.
`in compositions of this invention.
`Characteristically, the components comprising cor
`In addition to methionine, other stabilizing agents,
`nea storage media include an aqueous electrolyte solu
`such as albumin and edetate disodium (EDTA), can be
`tion (e.g. Gibco MEM or TC 199), a buffer system (e.g.,
`added to further enhance the stability of the ophthalmic
`bicarbonate plus hydroxyethyl piperidine ethanesul
`composition in addition to the beneficial effect of methi
`fonic acid, "HEPES") to maintain an essentially neutral
`onine. The amount of albumin can be added at concen
`pH of about 7.0 to about 7.4, an energy and carbon
`trations of about 1.0% w/v or less. The edetate diso
`Source (glucose and/or pyruvate), an antioxidant (gluta
`dium can be added at a concentration of about 0.1%
`thione or ascorbate), deturgescent (e.g., a polysaccha
`10
`w/v or less. The edietate disodium acts as a scavenger of
`ride such as carboxyalkyl cellulose, polyvinyl pyrrol
`metal ions known to catalyze many oxidation reactions,
`idone, polyvinyl alcohol, hyaluronic acid and especially
`thus providing an additional stabilizing agent.
`dextran sulfate), antibiotic (e.g., gentamicin, streptomy
`Purified water USP is added to the ophthalmic prepa
`cin and the like) and antimycotic (e.g., fungizone)
`ration to bring the composition to its final volume. Puri
`agents, membrane stabilizing agents (e.g. vitamins A
`fied or sterile water can be employed. The ophthalmic
`and B, retinoic acid and/or cofactors) and other op
`preparation then can be packaged in squeezable plastic
`tional components such as a glycosaminoglycan (Std. or
`containers made of low density polyethylene which is
`purified, low molecular weight A, B or Cheparin sul
`very permeable to oxygen. The added methionine inhib
`fate, keratan sulfate and/or dermatan sulfate; chondroi
`its the rapid oxidation of the methionine residue of the
`20
`tin sulfate (A, B or C isomer) or sodium hyaluronate),
`polypeptide which otherwise would occur, thus provid
`additional mitotic-enhancing agents, such as transferrin,
`ing a more stable ophthalmic preparation having a
`selenous acid and linoleic acid, preservatives, such as
`longer therapeutic life.
`the polymeric quaternary ammonium salts and the like.
`In another embodiment of the present invention, epi
`The storage media also contain essential nutrients and
`dermal growth factor, methionine and any of the fore
`25
`minerals in at least the minimum concentration required
`going optional pharmaceutical adjuvants can be dis
`for cell growth. In general, they contain inorganic salts,
`solved in a minimal amount of purified water U.S.P. or
`such as calcium, magnesium, iron, sodium and potas
`other sterile water and incorporated into a semisolid
`sium salts of carbonates, nitrates, phosphates, chloride
`dosage form, such as an ointment, cream or lotion, use
`and the like, essential and nonessential amino acids,
`ful for treating or preventing eye conditions according
`30
`vitamins and other essential nutrients. Chemically de
`to the methods as practiced in the art. As before, suffi
`fined basal nutrient media are commercially available,
`cient methionine (L, D or DL isomer) is added to the
`for example, from Gibco Laboratories (3175 Stanley
`preparation to inhibit the methionine residue(s) from
`Road, Grand Island, New York 14073) and Microbio
`being oxidized to methionine sulfoxide. Typically, epi
`logical Associates (P.O. Box 127, Biggs Ford Road,
`dermal growth factor is present in such compositions in
`35
`Walkersville, Maryland 21793) under the names Eagle's
`amounts of from about 0.001% w/w to about 0.01%
`minimal essential medium and TC 199, respectively.
`w/w of the semisolid preparation and the methionine is
`Eye tissue storage media have been adopted from these
`added in amounts of from about 0.01% w/w to about
`nutrient media. Commercially available cornea storage
`1.0% w/w. Preferably, the final concentration ranges
`media useful in the present invention include MK, Dex
`from about 0.01% w/w to about 0.3% w/w.
`solTM, Optisol TM and Optisol Plus TM (obtainable
`Typical emollients in which the foregoing aqueous
`from Chiron Ophthalmics, Irvine California).
`composition of methionine and polypeptide can be in
`The resulting cornea storage media are packaged in
`corporated include white petrolatum U.S.P., mineral
`20 ml glass containers. This makes for easy storage in
`oil, and vegetable oils, such as olive oil, cottonseed oil
`and almond oil, and the like. Optional pharmaceutical
`tissue banks as well as convenient application of the
`45
`adjuvants also can be added. The optional adjuvants can
`media to ocular tissue. The added methionine stabilizes
`the polypeptides in the medium from rapid oxidation,
`include surface active agents, such as sorbitans, polysor
`thus prolonging the effective life of the preparation.
`bates and the like; an antioxidant, such as butylated
`hydroxytoluene (BHT) and the like; solvents, such as a
`The invention is further illustrated by the following
`propylene glycol, glycerin and the like and suspending
`examples. Various modifications can be made without
`50
`departure from the spirit and scope of the invention.
`agents, such as magma of bentonite and the like. Emulsi
`fying agents, such as isopropyl myristate, lanolin alco
`Accordingly, it is not intended that the invention be
`limited except as by the intended claims.
`hol and the like also can be added to further stabilize the
`semisolid composition.
`Example I
`In still another embodiment of the present invention,
`Stability of Recombinant Human Epidermal Growth
`methionine can be added to tissue storage media, such
`as cornea storage medium, comprising growth factors
`Factor (rhEGF) Acetate Solutions with Methionine at
`having an amino acid sequence which comprises at least
`4 C.
`The following four formulations were prepared and
`one methionine residue. Sufficient methionine is added
`to the tissue storage media to inhibit the oxidation of the
`filled in individual 6 ml clear low density polyethylene
`methionine residue in the growth factors to methionine
`(LDPE) ophthalmic containers. Only 2 ml of each solu
`sulfoxide.
`tion were placed in each container to allow for suffi
`Typically, the growth factors are added to a cornea
`cient oxygen in the head space. A sufficient amount of
`storage medium in amounts generally ranging from
`0.05M phosphate buffered saline was added to each
`about 1 picomolar to about 10 micromolar. Methionine
`formulation to maintain a pH of from about 7.0 to about
`65
`(L, D or DL isomer) is characteristically added in
`7.4. The recombinant human epidermal growth factor
`amounts which range from about 0.001% w/v to about
`(rhEGF) acetate used was obtained from Chiron Corp.,
`0.005% w/v. Preferably, the concentration of methio
`Emeryville, California.
`
`60
`
`15
`
`55
`
`

`

`7
`
`Formulation I:
`
`Formulation II:
`
`Formulation III:
`
`Formulation IV:
`
`0.01% w/v rhEGF in 0.05M phosphate
`buffered saline with 0.1% w/v L
`methionine USP, and 0.05% w/v EDTA
`USP, qs with a sufficient amount of
`purified water USP.
`0.01% w/v rhEGF in 0.05M phosphate
`buffered saline with 0.1% w/v L
`methionine USP, and 0.1% w/v. EDTA
`USP, qs with a sufficient amount of
`purified water USP.
`0.01% w/v rhEGF in 0.05M phosphate
`buffered saline with 0.015% w/v
`EDTA USP.
`0.01% w/v rhEGF in 0.05M phosphate
`buffered saline with 0.1% w/v EDTA
`USP.
`
`5
`
`10
`
`15
`
`5,272,135
`8
`that the addition of L-methionine reduced the rate of
`oxidation of rhEGF drastically even at room tempera
`ture. Formulation I showed a 2.8% increase in oxidized
`rhEGF in 105 days while Formulation III (without
`L-methionine) showed a 16.5% increase in oxidized
`rhEGF over the same time period. FIG. 2 graphically
`presents the data in Table II. The addition of methio
`nine to a formulation containing rhEGF clearly stabi
`lized the rhEGF against oxidation.
`TABLE II
`Percent Oxidation of rhEGF Acetate
`Formulations I-IV
`at Room Temperature
`TIME (DAYS) FORM. I FORM, II FORM. III FORM. IV
`O
`.8%
`1.6%
`1.8%
`1.6%
`14
`2.5
`2.4
`24
`2.9
`2.9
`30
`17.3
`3.1
`2.6
`60
`8.3
`5.
`4.6
`105
`Formulation I: with 0.1% L-methionine, with 0.015% EDTA
`Formulation II: with 0.1% L-methionine, with 0.1% EDTA
`Formulation III: without L-methionine, with 0.05% EDTA
`Formulation IV: without L-methionine, with 0.1% EDTA
`
`12.5
`
`10.5
`
`1.3
`2.5
`
`25
`
`Each container was stored at 4' C. for a period of 105
`days, Samples were withdrawn from each formulation
`on day 0, 14, 24, 30, 60 and 105 and assayed in a high
`performance liquid chromatograph (HPLC) to deter
`20
`mine the percent oxidation of rhEGF to rhEGF methi
`onine sulfoxide. Table I shows the percent oxidation
`rates of rhEGF. FIG. 1 presents the data from Table I
`graphically. It is clear that the addition of L-methionine
`reduced the rate of oxidation of rhEGF drastically.
`Formulation I showed a 1.4% increase in oxidized
`rhEGF in 105 days while Formulation III (without
`L-methionine) showed 17.7% oxidation in 105 days.
`Increasing the level of EDTA from 0.01% w/v to 0.1%
`w/v did not appear to further enhance the stability of 30
`rhEGF in the presence of L-methionine, while it ap
`peared to enhance the stability of rhEGF in the absence
`of L-methionine.
`FIG. 3 and 4 show the HPLC chromatograms, mil
`liabsorbance units (mAU) versus time, of the initial and
`35
`aged (105 days) samples of Formulations I and III, re
`spectively. The rhEGF is labeled peak D and the
`rhEGF methionine sulfoxide is labeled peak C. Com
`parison of the chromatograms in FIGS. 3A, 3B and 4A,
`4B clearly shows that the addition of L-methionine 40
`reduced the amount of rhEGF oxidized to rhEGF me
`thionine sulfoxide. The amount of rhEGF that oxidized
`to rhEGF methionine sulfoxide as seen in FIG. 4B was
`considerably more than the amount of rhEGF that
`oxidized to rhEGF methionine sulfoxide in FIG. 3B. 45
`TABLE I
`Percent Oxidation of rhEGF Acetate
`Formulations I-IV
`at 4 C.
`TIME (DAYS) FORM. I FORM. II FORM, III FORM. IV 50
`0
`1.8%
`1.6%
`1.8%
`.6%
`14
`2.5
`2.4
`24
`4.1
`2.7
`30
`17.4
`3.2
`2.7
`60
`19.5
`3.6
`3.2
`105
`Formulation : with 0.1% L-methionine, with 0.05% EDTA
`Formulation II: with 0.1% L-methionine, with 0.1% EDTA
`Formulation III: without L-methionine, with 0.015%. EDTA
`Formulation IV: without L-methionine, with 0.1% EDTA
`
`Example III
`Recombinant Human Epidermal Growth Factor
`(rhEGF) Ophthalmic Solution, 0.01% w/v.
`The following ingredients were sterile filtered into
`sterile low density polyethylene (LDPE) ophthalmic
`containers to form a pharmaceutically stable and effec
`tive ophthalmic solution. The resulting ophthalmic so
`lutions were considerably more stable and had a longer
`therapeutic life than if the methionine had not been
`added. Table III discloses the results of the oxidation
`rates in two batches of a 0.01% EGF solution with
`methionine. Clearly, methionine reduced the rate of
`methionine oxidation of the EGF in both batches to an
`insignificant level.
`
`Ingredient
`rhEGF
`Sodiurn Chloride
`Sodium Phosphate
`Monobasic
`Monohydrate
`Sodium Phosphate
`Dibasic
`Heptahydrate
`Edetate Disodium
`(EDTA)
`L-Methionine
`Purified Water
`
`Concentration, percent w/v
`0.01
`0.55
`0.19
`
`USP
`USP
`
`USP
`
`USP
`
`USP
`USP
`
`0.96
`
`0.05
`
`0,10
`qs 100
`
`TABLE III
`0.01% w/v EGF Ophthalmic Solution
`Percent of EGF Oxidized at 4 C.
`0.0% w/v EGF Sol.
`0.0% w/v EGF Sol.
`with methionine
`with methionine
`Time
`Lot #IPL0001
`Lot #39-60
`(Days)
`1.02
`0.51
`Initial
`.10
`0.966
`30
`2.28
`1.72
`60
`2.9
`1,645
`200
`'Obtained from Chiron Corp., Emeryville, California.
`
`15
`
`8.7
`
`12.5
`13.1
`
`55
`
`60
`Example II
`Stability of Recombinant Human Epidermal Growth
`Factor (rhEGF) Acetate Solutions with Methionine at
`Room Temperature.
`Four formulations were prepared and tested as in 65
`Example I except that the formulations were stored at
`room temperature (23'-2' C), Table II shows the
`percent oxidation rates at room temperature. It is clear
`
`

`

`Concentration, percent w/v
`0.003
`0.55
`0.9
`
`USP
`USP
`
`Ingredient
`rhEGF
`Sodium Chloride
`Sodium Phosphate
`Monobasic
`Monohydrate
`Sodium Phosphate
`Dibasic
`Heptahydrate
`USP
`L-Methionine
`USP
`Purified Water
`Obtained from Chiron Corp., Emeryville, California.
`
`USP
`
`0.96
`
`0.10
`qs 100
`
`15
`
`20
`
`25
`
`5,272,135
`10
`tion during the period in which the composition will be
`stored and used.
`4. The method according to claim 1, wherein the
`polypeptide is a growth factor.
`5. The method according to claim 4, wherein the
`growth factor comprises one of epidermal growth fac
`tor, insulin-like growth factor I, nerve growth factor,
`transforming growth factor alpha precursor, transform
`ing growth factor beta, transforming growth factor beta
`precursor, fibroblast growth factor, vaccinia growth
`factor, platelet derived growth factor, or a methionine
`containing biologically active fragment, precursor or
`analog of one of these growth factors.
`6. The method according to claim 5, wherein the
`growth factor comprises bovine, porcine, rat, mouse or
`human growth factor.
`7. The method according to claim 5, wherein the
`growth factor comprises natural or recombinant
`growth factor.
`8. The method according to claim 5, wherein the
`composition is a liquid, the growth factor comprises
`epidermal growth factor in a sufficient amount such
`that its concentration ranges from about 0.001% w/v to
`about 0.1% w/v and the methionine is added such that
`its concentration ranges from about 0.01% w/v to
`about 0.3% w/v.
`9. The method according to claim 5, wherein the
`composition is in the form of a semi-solid, the growth
`factor comprises epidermal growth factor in a sufficient
`amount such that its concentration ranges from about
`0.001% w/w to about 0.1% w/w and the methionine is
`added such that its concentration ranges from about
`0.0% w/w to about 0.3% w/w.
`10. The method according to claim 1, wherein the
`liquid or semi-solid composition further comprises one
`or more pharmaceutically acceptable adjuvants.
`11. The method according to claim 10, wherein the
`composition is a liquid and the pharmaceutically accept
`able adjuvants comprise one or more of sodium chlo
`ride, phosphates, citrates, acetates, lactates, edietate
`disodium, povidone, polyvinyl alcohol, glycerin, so
`dium carboxymethyl cellulose, hydroxyproprylmethyl
`cellulose, sodium hydroxide, hydrochloric acid, acetic
`acid, albumin, or quaternary ammonium salts.
`12. The method according of claim 10, wherein the
`composition is a semi-solid and the pharmaceutically
`acceptable adjuvants comprise one or more of white
`petrolatum, mineral oil, vegetable oils, sorbitans, poly
`sorbates, butylated hydroxytoluene, propylene glycol,
`glycerin, magma of bentonite, isopropyl myristate, lan
`olin alcohol or edetate disodium.
`13. A stable liquid or semi-solid pharmaceutical or
`therapeutic preparation which comprises a polypeptide
`having an amino acid sequence which compiles at least
`one methionine residue and methionine, wherein the
`methionine is present at a concentration sufficient to
`inhibit oxidation of the at least one methionine residue
`of the polypeptide, said concentration being between
`about 0.01% w/v and 0.3% w/v.
`14. A stable liquid pharmaceutical preparation in
`accordance with claim 13, wherein the liquid prepara
`tion is an aqueous ophthalmic solution comprising an
`epidermal growth factor at a concentration range of
`from about 0.00% w/v to about 0.10% w/v and fur
`ther comprising methionine at a concentration range of
`from about 0.01% w/v to about 0.3% w/v.
`15. A stable semi-solid pharmaceutical preparation in
`accordance with claim 13, wherein the semi-solid prep
`
`9
`Example IV
`Recombinant Human Epidermal Growth Factor
`(rhEGF) Ophthalmic Solution, 0.003% w/v.
`The following ingredients were sterile filtered into a 5
`low density polyethylene (LDPE) ophthalmic bottle to
`form a pharmaceutically stable and effective ophthal
`mic solution. The resulting ophthalmic solution was
`considerably more stable and had a longer therapeutic
`life than if the methionine had not been added. After 95
`days storage at 4' C. the percent of EGF oxidized in
`creased from 2.55 to 3.88, an increase of only 1.33 per
`cent.
`
`10
`
`Example V
`Recombinant Human Epidermal Growth Factor so
`(rhEGF) Ophthalmic Ointment, 0.01% w/w.
`The following ingredients are combined to form a
`pharmaceutically stable and effect