Ulllted States Patent [19]
`Yamazaki et al.
`
`US006120761A
`[11] Patent Number:
`[45] Date of Patent:
`
`6,120,761
`Sep. 19, 2000
`
`[54] ERYTHROPOIETIN SOLUTION
`PREPARATION
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`[75] Inventors: Tadao Yamazaki; Toshiari Morita;
`Hiroshi Na ai all of Tok 0 Ja an
`g ’
`y ’
`p
`[73] Assignee; Chugai Seiyaku Kabushiki Kaisha,
`Tokyo, Japan
`
`4,877,608 10/1989 Lee et al. ............................. .. 424/858
`4,992,419
`2/1991 Woog et a1.
`514/8
`1
`5 580 856 12/1996 P t
`1 k‘ t
`514/21
`
`
`, , 5,691,312 11/1997 Paques .................................... .. 514/12 resres1ea..
`
`FOREIGN PATENT DOCUMENTS
`
`[21] Appl. No.:
`[22] PCT Filed:
`.
`[86] PCT No..
`§ 371 Date:
`
`09/171,737
`Apr. 25, 1997
`
`PCT/JP97/01449
`Dec. 16, 1998
`
`§ 102(6) Date: DEC. 16, 1998
`
`NO.Z
`PCT Pub. Date: Nov. 6, 1997
`
`61-97229
`
`5/1986 Japan .
`j
`64-42442 2/1989 Japan .
`64_71818 3/1989 Japan '
`3-044333
`2/1991 Japan ~
`3-170437 7/1991 Japan .
`4-108737 4/1992 Japan .
`93/03744 3/1993 WIPO .
`96/17593
`6/1996 WIPO _
`9608143 9/1996 WIPO '
`
`_
`_
`_
`_
`_
`Forelgn Apphcatlon Pnonty Data
`[30]
`Apr. 26, 1996
`[JP]
`Japan .................................. .. 8-131226
`Oct. 30, 1996
`[JP]
`Japan
`.. 8-303956
`
`Primary Examiner—Jeffrey E. Russel
`Attorney, Agent, or Firm—Morgan & Finnegan, L.L.P.
`[57]
`ABSTRACT
`
`7
`
`.
`
`" A61K 38/19’ A61K 38/22
`[51] Int‘ Cl‘ "
`[52] US. Cl. ................................. .. 424/85.1; 514/21
`[58] Field of Search ................................... .. 530/350, 351,
`530/397; 514/8, 12, 21; 424/85.1
`
`E3
`2
`m
`
`E1 8
`
`i 100
`
`This invention provides an erythropoietin solution prepara
`tion containing an amino acid as a stabilizer, and having
`excellent 1Ong_term Storage Stability‘
`
`11 Claims, 4 Drawing Sheets
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`
`L-HISTIDINE HYDROCHLORIDE CONCENTRATION (mg/ml)
`
`CFAD Exhibit 1006
`
`1
`
`

`

`U.S. Patent
`
`Sep. 19,2000
`
`Sheet 1 of4
`
`6,120,761
`
`
`
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`L—ARGININE HYDROCHLORIDE CONCENTRATION (mg/ml)
`
`2
`
`

`

`U.S. Patent
`
`Sep. 19,2000
`
`Sheet 2 of4
`
`6,120,761
`
`F 1'9. 2
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`
`L-LYSINE HYDROCHLORIDE CONCENTRATION (mg/ml)
`
`3
`
`

`

`U.S. Patent
`
`Sep. 19,2000
`
`Sheet 3 of4
`
`6,120,761
`
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`
`L-HISTIDINE HYDROCHLORIDE CONCENTRATION (mg/ml)
`
`4
`
`

`

`U.S. Patent
`
`Sep. 19, 2000
`
`Sheet 4 of4
`
`6,120,761
`
`i234i'67 97/0
`
`5
`
`

`

`1
`ERYTHROPOIETIN SOLUTION
`PREPARATION
`
`TECHNICAL FIELD
`
`This invention relates to an erythropoietin solution prepa
`ration.
`
`BACKGROUND ART
`
`Erythropoietin (hereinafter referred to as EPO) is an
`acidic glycoprotein hormone Which promotes the differen
`tiation and proliferation of erythroid progenitor cells. This
`hormone is secreted chie?y by the kidney. Erythrocytes are
`present abundantly in the blood for certain periods, and are
`then destroyed by the spleen, etc. (their mean life in humans
`is about 120 days). HoWever, red blood cells are constantly
`supplied from the bone marrow, so that the peripheral total
`erythrocyte count is kept constant in a normal state. EPO
`plays a central role in maintaining such homeostasis of
`erythrocytes in the living organism.
`High purity human urinary EPO Was obtained by puri?
`cation from a large volume of urine from patients With a
`plastic anemia. This enabled cloning of human EPO gene.
`NoWadays, it has become possible to produce a large amount
`of recombinant human EPO in animal cells by genetic
`engineering technology. The applicant of this invention has
`succeeded in producing a preparation (lyophiliZed
`preparation) of the puri?ed EPO, and supplies it to the
`market in the form of renal anemia alleviating agents and so
`on.
`Drug design for supplying the market With stable EPO
`preparations requires that chemical changes (hydrolysis,
`disul?de exchange reaction, etc.) or physical changes
`(denaturation, agglutination, adsorption, etc.) observed With
`EPO be suppressed. Products noW on the market contain
`human serum albumin or puri?ed gelatin Which is generally
`used as a stabiliZer. These substances have been added in
`these products to suppress chemical or physical changes.
`Since human serum albumin is a blood product relying on
`donated blood for its supply, the necessity for its addition
`has been questioned. Regarding the addition of a protein
`other than the albumin or gelatin as a stabiliZer, it is dif?cult
`to avoid the risk of viral contamination completely.
`Peptide drugs are often lyophiliZed for stabiliZation.
`HoWever, lyophiliZation increases manufacturing costs, and
`involves an increased risk due to mechanical troubles.
`For the foregoing reasons, demand is groWing for an EPO
`preparation as an alternative to a lyophiliZed preparation, the
`EPO preparation being free from inclusion of a protein as a
`stabiliZer, and stable during long-term storage.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`DISCLOSURE OF THE INVENTION
`To satisfy the above demand, We, the inventors, have
`conducted extensive studies. As a result, We have found that
`EPO can be converted into a stable EPO solution preparation
`free from human serum albumin and puri?ed gelatin by
`adding a certain amino acid as a stabiliZer. This ?nding has
`led us to complete the present invention.
`That is, the present invention provides an erythropoietin
`solution preparation containing an amino acid as a stabiliZ
`ing agent or stabiliZer.
`“To stabiliZe; stabiliZing” in this speci?cation refers to
`storing, or the erythropoietin solution preparation, for
`example, for more than 2 years at 10° C., or for more than
`6 months at 25° C., or for more than 2 Weeks at 40° C. While
`keeping the residual rate of erythropoietin at 90% or higher,
`preferably 95% or higher, more preferably 98% or higher.
`
`55
`
`60
`
`65
`
`6,120,761
`
`2
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 is a graph shoWing the relation betWeen the
`concentration of L-arginine hydrochloride and the residual
`rate of erythropoietin;
`FIG. 2 is a graph shoWing the relation betWeen the
`concentration of L-lysine hydrochloride and the residual rate
`of erythropoietin;
`FIG. 3 is a graph shoWing the relation betWeen the
`concentration of L-histidine hydrochloride and the residual
`rate of erythropoietin; and
`FIG. 4 shoWs an SDS-polyacrylamide gel electrophoresis
`pattern illustrating the degradation product suppressing
`effect of preparations to Which various amino acids have
`been added (an electrophoretogram), in Which lane 1:
`molecular Weight marker, lane 2: amino acid-free
`preparation, lane 3: L-leucine-containing preparation, lane
`4: 1-phenylalanine-containing preparation; lane 5:
`L-tryptophan-containing preparation, lane 6: L-serine
`containing preparation, lane 7: L-cysteine-containing
`preparation, lane 8: monosodium L-glutamate monohydrate
`containing preparation, lane 9: L-arginine hydrochloride
`containing preparation, and lane 10: L-histidine
`hydrochloride-containing preparation.
`
`BEST MODE FOR CARRYING OUT THE
`INVENTION
`
`EPO for use in the solution preparation of the present
`invention has substantially the same biological activity as
`that of mammalian, especially, human EPO, and includes
`naturally occurring EPO and EPO obtained by genetic
`recombination. EPO from genetic recombination includes
`EPO having the same amino acid sequence as that of
`naturally occurring EPO, or EPO With this amino acid
`sequence from Which one or more of the amino acids have
`been deleted, or in Which one or more of the amino acids
`have been substituted, or to Which one or more amino acids
`have been added, and Which, hoWever, retains the above
`mentioned biological activity. The EPO in the present inven
`tion may be produced by any methods, for example, a
`method comprising extraction from human urine, folloWed
`by separation and puri?cation, in various manners; and a
`method involving production in E. coli, yeast, or Chinese
`hamster ovary cells, folloWed by extraction, separation and
`puri?cation in various manners.
`The amino acid added as a stabiliZer in the present
`invention includes free amino acids, and their salts such as
`sodium salts, potassium salts and hydrochlorides. The solu
`tion preparation of the present invention may have one or
`more of these amino acids added in combination. The
`preferred amino acids are D-, L- and DL-forms of leucine,
`tryptophan, serine, glutamic acid, arginine, histidine and
`lysine, and their salts. More preferable are L-leucine,
`L-tryptophan, L-glutamic acid, L-arginine, L-histidine and
`L-lysine, and their salts. Particularly preferable are
`L-arginine, L-histidine and L-lysine, and their salts. Most
`preferable are L-histidine and its salts.
`The solution preparation of the present invention,
`preferably, is substantially free from protein as a stabiliZer.
`For the amount of the amino acid added to the solution
`preparation of the present invention, a preferred range can be
`set by a testing method (to be described later on) depending
`on the type of the amino acid used. Generally, the amount of
`the amino acid added is 0.001 to 50 mg/ml, but preferably
`0.1 to 40 mg/ml, more preferably 1 to 10 mg/ml for arginine,
`preferably 0.5 to 10 mg/ml, more preferably 1 to 10 mg/ml
`
`6
`
`

`

`6,120,761
`
`3
`for lysine, preferably 0.5 to 10 mg/ml, more preferably 1.0
`to 4.0 mg/ml, and most preferably 1.0 to 2.0 mg/ml for
`histidine. As Will be described later on, the highest residual
`rate of EPO Was obtained When L-arginine hydrochloride
`and L-lysine hydrochloride Were each added in an amount of
`about 1 to 5 mg/ml as free amino acid, or When L-histidine
`hydrochloride Was added in an amount, as free amino acid,
`of 1 to 10 mg/ml in an accelerated testing performed for 2
`Weeks at 40° C., or 0.5 to 5 mg/ml in a 25° C.-6 month
`accelerated testing.
`The amount of EPO contained in the solution preparation
`of the present invention can be determined according to the
`type of disease to be treated, the severity of the disease, the
`age of the patient, and so forth. Generally, its amount is 100
`to 500,000 IU/ml, preferably 200 to 100,000 IU/ml, more
`preferably 750 to 72,000 IU/ml. The solution preparation of
`the present invention is administered usually by a parenteral
`route, for example, by injection (subcutaneous or
`intravenous), or percutaneously, transmucosally or
`transnasally, but oral administration is also possible.
`The solution preparation of the present invention may
`contain, in addition to EPO and the amino acid, ingredients
`usually added to a preparation in the form of a solution, such
`as polyethylene glycol; sugars, e.g., dextran, mannitol,
`sorbitol, inositol, glucose, fructose, lactose, xylose,
`mannose, maltose, sucrose, and raf?nose; inorganic salts,
`e.g., sodium chloride, potassium chloride, calcium chloride,
`sodium phosphate, potassium phosphate, and sodium hydro
`gen carbonate; organic salts, e.g., sodium citrate, potassium
`citrate and sodium acetate; and, if desired, sulfur-containing
`reducing agents, e.g., glutathione, thioctic acid, sodium
`thioglycolate, thioglycerol, ot-momothioglycerol and
`sodium thiosulfate. The preferred salt is sodium chloride. It
`is also preferred to add an adsorption preventing agent, such
`as a polyoxyethylene sorbitan alkyl ester, to the solution
`preparation of the present invention. Particularly preferable
`polyoxyethylene sorbitan alkyl esters are polysorbate 20, 21,
`40, 60, 65, 80, 81 and 85, and most preferably, polysorbate
`20 and/or 80. The preferred amount of polysorbate 20 and/or
`80 added is 0.01 to 1 mg/ml, more preferably 0.05 to 0.1
`mg/ml.
`The solution preparation of the present invention can be
`prepared by dissolving the above-mentioned components in
`an aqueous buffer publicly knoWn in the ?eld of solution
`preparations, such as phosphate and/or citrate buffer. The
`preferred phosphate buffer is a sodium monohydrogen
`phosphate-sodium dihydrogen phosphate buffer, While the
`preferred citrate buffer is a sodium citrate buffer. The pH of
`the solution preparation of the present invention is 5.0 to 8.0,
`preferably, 6.0 to 7.0.
`Japanese Unexamined Patent Publication No. 64-71818
`discloses a human protein preparation characteriZed by
`containing urea, an amino acid, and a nonionic Wetting
`agent. HoWever, the solution preparation of the present
`invention preferably does not contain urea, because it is not
`clear Whether urea contributes to the long-term stabiliZation
`of a glycoprotein such as erythropoietin. Areaction betWeen
`urea degradation products and protein is also knoWn to take
`place (Protein Chemistry 3, Kyoritsu Shuppan, Chapter 12),
`Which may adversely affect the preparation. Furthermore,
`the feWer ingredients added to the preparation, the better the
`results that can be expected.
`The solution preparation of the present invention is usu
`ally contained in a sealed, steriliZed plastic or glass con
`tainer. The solution preparation can be supplied as a pre
`scribed dose in an ampoule, vial or disposable syringe, or in
`a multiple dose form such as a bag or bottle for injection.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`55
`
`60
`
`65
`
`4
`EPO solution preparations containing various amino acids
`Were prepared, and subjected to an accelerated testing con
`ducted for 2 Weeks at 40° C. The EPO content in each of the
`preparations after the test Was measured by RP-HPLC
`(reversed phase high performance liquid chromatography)
`to investigate the effect of amino acid addition on this
`content. As a result, the residual rate of EPO Was found to
`be higher in the solution preparations containing L-leucine,
`L-tryptophan, monosodium L-glutamate monohydrate,
`L-arginine hydrochloride, L-histidine hydrochloride, and
`L-lysine hydrochloride than in the solution preparations
`containing no amino acids. The results of SDS
`polyacrylamide gel electrophoresis demonstrated L-arginine
`hydrochloride and L-histidine hydrochloride to be effective
`in suppressing the formation of EPO degradation products to
`be observed in the preparation after the accelerated testing.
`Of the amino acids thus shoWn to be effective When
`added, L-arginine hydrochloride, L-lysine hydrochloride
`and L-histidine hydrochloride Were examined for the effect
`of their concentrations on the stabiliZation of the prepara
`tion. That is, EPO preparations to Which L-arginine
`hydrochloride, L-lysine hydrochloride or L-histidine hydro
`chloride Was added in various concentrations Were made,
`and a 40° C.-2 Week accelerated testing Was conducted on
`these preparations. Upon completion of the test, the residual
`rates of EPO in the preparations tended to peak at concen
`trations of about 1 to 5 mg/ml in the case of L-arginine
`hydrochloride and L-lysine hydrochloride. With L-histidine
`hydrochloride, maximum EPO residual rate Was achieved at
`a concentration of 1 to 10 mg/ml. A 25° C.-6 month
`accelerated testing Was also performed in EPO preparations
`to Which L-histidine hydrochloride Was added in various
`concentrations. The EPO residual rate Was maximal at the
`concentrations of 0.5 to 5 mg/ml. These ?ndings shoWed
`L-arginine hydrochloride, L-lysine hydrochloride, and
`L-histidine hydrochloride to have the optimum concentra
`tion of addition.
`The EPO solution preparation of the present invention is
`a safe preparation free from foreign proteins such as human
`serum albumin or puri?ed gelatin, and Without the risk of
`viral contamination. The amino acid added thereto is
`cheaper than these conventional stabiliZers, and the cost
`incurred during the manufacturing process is also loWer than
`that for a lyophiliZed product. Thus, the preparation of this
`invention is advantageous economically. Furthermore, the
`solution preparation of the present invention need not be
`dissolved in a buffer, but can be used as it is. This lessens
`labor in using it in comparison With a lyophiliZed prepara
`tion. Because of these various advantages, the industrial
`applicability of the present invention is great.
`The present invention Will noW be described in further
`detail by reference to the folloWing examples, but its scope
`is not restricted thereby.
`
`EXAMPLES
`
`Testing Method
`A 5 ml glass vial Was charged With 1 ml of a dispensing
`solution containing the folloWing components/ml and being
`adjusted to pH 6.0 With a 10 mM phosphate buffer (Wako
`Pure Chemical Industries, Ltd.):
`
`EPO
`Nonionic surfactant
`(polysorbate 80, Nikko
`
`1,500 IU
`0.05 mg
`
`7
`
`

`

`-continued
`
`EPO
`Chemical Co., Ltd.)
`Sodium chloride
`Amino acid (Sigma Chemical
`Comp any)
`
`1,500 IU
`
`8.5 mg
`0 to 40 mg
`
`The ?lled vial Was stoppered, sealed, and used as a solution
`preparation. As an accelerated testing, the preparation Was
`alloWed to stand for 2 Weeks in a thermostatic chamber at
`40° C. Then, the preparation Was evaluated by RP-HPLC
`analysis (WATERS) and SDS-polyacrylamide gel electro
`phoresis analysis.
`
`Amino acid
`
`10
`
`Not added
`L-arginine
`hydrochloride
`
`15
`
`6,120,761
`
`6
`
`TABLE 2
`
`EPO residual rates after accelerated testing of
`L-arginine hydrochloride-containing
`preparations
`
`Amount added
`(mg/ml)
`
`EPO residual rate after 400 C.—2
`Week accelerated testing
`(% of initial content)
`
`0
`0.1
`1
`5
`10
`20
`40
`
`89.6
`92.7
`96.7
`96.1
`93.6
`92.0
`91.6
`
`Example 1
`
`Effect of the Addition of Various Amino Acids on EPO
`Residual Rate
`
`20
`
`In accordance With the foregoing testing method, the
`solution preparations containing various amino acids tabu
`lated beloW Were produced, and subjected to the 40° C.—2
`Week accelerated testing. Then, their EPO residual rates
`Were determined by the RP-HPLC method. The results are
`shoWn in Table 1.
`
`TABLE 1
`
`EPO residual rates after accelerated testing
`of various amino acids-containing solution
`preparations
`
`Amount added
`(mg/ml)
`
`EPO residual rate after
`400 C.—2 Week accelerated
`testing
`(% of initial content)
`
`0
`10
`10
`5
`10
`10
`10
`
`10
`
`10
`
`10
`
`83.9
`91.6
`57.8
`97.0
`85.2
`47.1
`93.9
`
`93.6
`
`99.7
`
`95.8
`
`Amino acid
`
`Not added
`L-leucine
`L-phenylalanine
`L-tryptophan
`L-serine
`L-cysteine
`Monosodium
`L-glutamate
`monohydrate
`L-arginine
`hydrochloride
`L-histidine
`hydrochloride
`L-lysine
`hydrochloride
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`As shoWn above, L-leucine, L-tryptophan, monosodium
`L-glutamate monohydrate, L-arginine hydrochloride,
`L-histidine hydrochloride, and L-lysine hydrochloride led to
`particularly marked EPO residual rates.
`
`55
`
`Example 2
`
`Effect of the Addition of an Amino Acid in Various Con
`centrations on EPO Residual Rate
`In accordance With the foregoing testing method, EPO
`solution preparations containing L-arginine hydrochloride in
`various concentrations indicated beloW Were produced, and
`subjected to the same 40° C.—2 Week accelerated testing.
`Then, their EPO residual rates Were determined by the
`RP-HPLC method. The results are shoWn in Table 2.
`
`60
`
`65
`
`The above results are depicted as a graph in FIG. 1.
`As shoWn above, L-arginine hydrochloride led to maxi
`mum EPO residual rates in a concentration range of about 1
`to 5 mg/ml.
`Then, the same test Was conducted using L-lysine hydro
`chloride. The amounts of L-lysine hydrochloride added and
`the EPO residual rates after the accelerated testing are
`shoWn in Table 3.
`
`TABLE 3
`
`EPO residual rates after accelerated testing of
`L-lysine hydrochloride-containing preparations
`
`Amino acid
`
`Not added
`L-lysine
`hydrochloride
`
`Amount added
`(mg/ml)
`
`EPO residual rate after 400 C.—2
`Week accelerated testing
`(% of initial content)
`
`0
`0.5
`1
`5
`10
`
`88.7
`93.1
`95.8
`96.3
`90.2
`
`The above results are depicted as a graph in FIG. 2.
`As shoWn above, L-lysine hydrochloride also led to
`maximum EPO residual rates in a concentration range of
`about 1 to 5 mg/ml.
`Then, the same test Was conducted using L-histidine
`hydrochloride. The amounts of L-histidine hydrochloride
`added and the EPO residual rates after the accelerated
`testing are shoWn in Table 4.
`
`TABLE 4
`
`EPO residual rates after accelerated testing
`of L-histidine hydrochloride-containing
`preparations
`
`Amino acid
`
`Not added
`L-histidine
`hydrochloride
`
`Amount added
`(mg/ml)
`
`EPO residual rate after 400 C.—2
`Week accelerated testing
`(% of initial content)
`
`0
`0.5
`1
`5
`10
`
`91.5
`95.5
`97.3
`98.1
`99.7
`
`The above results are depicted as a graph in FIG. 3.
`As shoWn above, L-histidine hydrochloride led to maxi
`mum EPO residual rates in a concentration range of about 1
`to 10 mg/ml.
`In accordance With the aforementioned testing method,
`EPO solution preparations containing L-histidine hydro
`chloride in various concentrations indicated beloW Were
`
`8
`
`

`

`7
`produced, and subjected to a 25° C.-6 month accelerated
`testing. Then, their EPO residual rates Were determined by
`the RP-HPLC method. The results are shoWn in Table 5.
`
`6,120,761
`
`8
`25 mA constant current/gel
`3) Staining method (Western blotting)
`The electrophoresed gel Was transferred to a polyvi
`nylidene di?uoride membrane. Then, anti-EPO rabbit
`antiserum, biotin-labeled anti-rabbit IgG goat antibody, and
`biotinylated horseradish peroxidase Were used for color
`development With 3,3‘ -diaminobenZidine-hydrogen perox
`ide as a substrate.
`4) Results
`The results obtained are shoWn in FIG. 4. Compared With
`the amino acid-free preparation (lane 2), the monosodium
`L-glutamate monohydrate-containing preparation (lane 8),
`the L-arginine hydrochloride-containing preparation (lane
`9), and the L-histidine hydrochloride-containing preparation
`(lane 10) shoWed the marked effect of suppressing the
`formation of EPO degradation products.
`What is claimed is:
`1. An erythropoietin solution preparation containing
`erythropoietin in combination With L-histidine as a
`stabiliZer, Wherein the concentration of the L-histidine is 1.0
`to 5 mg/ml.
`2. An erythropoietin solution preparation containing
`erythropoietin in combination With L-histidine as a
`stabiliZer, Wherein the concentration of the L-histidine is 0.5
`to 5 mg/ml, and said solution preparation is dissolved in a
`phosphate buffer, a citrate buffer, or combination thereof.
`3. An erythropoietin solution preparation containing
`erythropoietin in combination With L-histidine as a
`stabiliZer, Wherein the concentration of the L-histidine is 0.5
`to 5 mg/ml, and Wherein the preparation does not contain
`L-lysine.
`4. The solution preparation of claim 1, 2 or 3, Which
`further contains a surfactant.
`5. The solution preparation of claim 4, Wherein the
`surfactant is a polyoxyethylene sorbitan alkyl ester.
`6. The solution preparation of claim 5, Wherein the
`surfactant is polysorbate 20 and/or 80.
`7. The solution preparation of claim 1, 2 or 3, Which
`further contains a salt.
`8. The solution preparation of claim 7, Wherein the salt is
`sodium chloride.
`9. The solution preparation of claim 1 or 3, Which has
`been dissolved in a buffer.
`10. The solution preparation of claim 9, Wherein the buffer
`is a phosphate buffer, a citrate buffer, or combination thereof.
`11. The solution preparation of claim 1, 2 or 3, Which does
`not contain a protein as a stabiliZer.
`
`*
`
`*
`
`*
`
`*
`
`*
`
`15
`
`25
`
`30
`
`35
`
`40
`
`TABLE 5
`
`EPO residual rates after accelerated testing of
`L-histidine hydrochloride-containing
`preparations
`
`Amount added
`(mg/ml)
`
`EPO residual rate after 25° C.—6
`month accelerated testing
`(% of initial content)
`
`10
`
`Amino acid
`
`Not added
`L-histidine
`hydrochioride
`
`0
`0.5
`1
`5
`10
`
`93.2
`99.3
`99.9
`97.9
`94.1
`
`As shoWn above, L-histidine hydrochloride led to maxi
`mum EPO residual rates in a concentration range of 0.5 to
`5 mg/ml, especially, at a concentration of 1 mg/ml.
`
`Example 3
`Effect of the Addition of Various Amino Acids on EPO
`Degradation Products
`In accordance With the aforementioned testing method,
`EPO solution preparations containing various amino acids
`Were produced, and subjected to a 40° C.-2 Week accelerated
`testing. Then, the formation of EPO degradation products
`Was investigated by the SDS-polyacrylamide gel electro
`phoresis analysis method.
`1) Preparation of Sample
`After the accelerated testing, a 1M TRIS-hydrochloride
`buffer (pH 6.8) containing SDS, glycerin, and Bromophenol
`Blue Was added to each of the EPO solution preparations
`containing each of the various amino acids indicated in
`Table 1 of Example 1. The mixture Was heated for 15
`minutes at 60° C. for use as a sample solution.
`2) Electrophoresis
`The sample solution (10 pl) Was electrophoresed under
`the folloWing operating conditions:
`a) Equipment: Slab gel electrophoresis apparatus (Bio-Rad
`Laboratories)
`b) Electrophoresis gel:
`SDS-PAGEmini8-16 (concentration gradient gel in poly
`acrylamide concentrations of 8 to 16%, Tefco)
`c) Electrophoresis temperature: 250 C.
`d) Electrophoresis conditions:
`
`9
`
`