WO1986000531A1 - Gamma-interferon composition - Google Patents

Abstract

Human gamma-interferon composition containing substantially no inorganic salts and being frozen or freeze-dried in the presence of amino acids. An aqueous human gamma-interferon solution containing substantially no inorganic salts is prepared by using an inorganic salt-free buffer as a buffer to be used in the final chromatographic procedure or by removing inorganic salts from a purified aqueous solution. This aqueous solution can be frozen or freeze-dried in the presence of an amino acid with extremely small reduction of the interferon activity, and the resulting composition has good storage stability and, when re-dissolved, forms no tubidity, thus being advantageously used as an antiviral agent, an antineoplastic agent, etc.

Description

 ' Specification

 Γ-type interferon composition

Technical field

 The present invention relates to a human r-type interferon composition.

 Background technology

 Human interferon is currently classified into three types: type a, type 3 and r. <Type 9 and type 9 interferon are relatively stable, are provided to the clinic mainly in the form of parenteral administration, and are undergoing systematic clinical research. On the other hand, r-type interferon (FN-sometimes abbreviated as r) is extremely unstable, and its activity is easily reduced during storage, freezing or freeze-drying operations of aqueous solutions, In addition, there is a problem that turbidity is observed in a solution obtained by re-dissolving the lyophilized product, and it has been extremely difficult to obtain a stable composition that can be used clinically. Therefore, it has a remarkably strong anti-inoles effect and anti-tumor effect [Salvin et al., Journal of Cancer Research Institute, Vol. 55, 123, pp. 1975]. It is a major obstacle to the clinical use of IFN-r, which is the most promising drug.

By the way, the drug substance of ら れ る-ら れ る ら れ る ら れ る 純度 製 剤 の も の 製 剤 に 製 剤 製 剤 製 剤 製 剤 製 剤 製 剤 製 剤 製 剤 製 剤 通常 通常 製 剤 製 剤 製 剤 製 剤 通常 通常 通常 通常 通常 通常 通常 通常 通常 通常 通常 通常 通常 通常 通常 通常. In this separation / purification process, various inorganic and organic compounds are used. For example, inorganic ions used for adjusting pH and ionic strength are also present in the purified aqueous solution of interfering solution. Was considered to act as a stabilizing agent and the like even when formulated. Running For example, it has been disclosed that the addition of an inorganic salt stabilizes interferon having no 耱 chain] (Japanese Patent Application Laid-Open No. 59-25364). .

Under such technical background, the present inventors surprisingly formulate using an aqueous IFN- _r solution with a reduced inorganic salt concentration, and surprisingly, in the freezing and freeze-drying ft crop, a conventional IFN-r aqueous solution containing an inorganic salt was used. As compared with the above operation, a stable IFN-r composition can be obtained in which the decrease in the IFN-r activity of the ^ layer is less and no turbidity is observed in the solution obtained by re-dissolving the obtained composition. The present inventors have further studied and completed the present invention.

 Disclosure of the invention

 An object of the present invention is to provide a human r-type interfering mouth composition which has been frozen or freeze-dried under conditions in which substantially no inorganic salt is present and amino acids coexist.

IFN-r used in the present invention may be any of IFN-r that is derived from human or natural or obtained by genetic recombination technology. In particular, an aqueous solution having a high concentration of human IFN- _r (rIFN-) obtained by genetic recombination technology is advantageously used.

The specific activity of human IFN-r is preferably 1 × 10 ⁵ to 1 × 10 ⁷ international units / ^ (I u ^), and 1 × 10 ² to 1 1 0 ⁷ I UZ, which has a ^{1 X 1 0 4 ~ 1 X} 1 0 7 IU ^ activity divided and are preferred.

As the above IFN-r aqueous solution, one containing substantially no inorganic salt is used. Here, the concentration of the inorganic salt may be 0.1 M or less, and is 0.05 5 or less. Is preferred. In addition, the inorganic ion strength may be such that the ion strength calculated from the inorganic ions is 0.1 or less. 0.05 or less, and! The reason is preferably 0.01 or less. Further, in the freeze-dried composition, the inorganic salt may be 30% or less, preferably 15% or less, and preferably 0.3% or less based on the total weight.

 The FN-Γ7_Κ solution that substantially contains inorganic salts can be prepared by using a buffer solution that does not contain inorganic salts as the buffer used in the purification step of IF-r and in the final chromatographic operation. ], Or by removing inorganic salts from the purified aqueous solution of IFN-r]).

 Preferred amino acids are glycine, na-alanin, | 3-alanine, oral isin, glutamic acid, and aspartic acid. Mono-amino aliphatic amino acids such as glycine are preferred, and glycine is particularly preferred. . Further, a physiologically acceptable salt or derivative thereof may be used. One or more of these amino acids can be used.The amino acids to be used can be commercially available, but in order to apply this composition to clinical applications, it should be of a quality sufficient for parenteral administration. Are preferred.

 It is preferable that the total amount of amino acids is 1 or more, preferably 5 to 50, equivalent to an IFN-r aqueous solution.

Further, the composition of the present invention may contain a reducing sulfur compound. Examples of the reducing sulfur compound include gnoletathione (reduced form), thioctic acid, thiocyanate diglycolone, thiocyanate ethanoamine, monothiocyanate glycerone, thiocyanate slate and carbon number ι. And thioalkanoic acids of from 7 to 7]. When a reducing sulfur compound is allowed to coexist, the IFN- _r aqueous solution 1D reducing sulfur compound is preferably 0.1 ^ or more, and 0.5 to 10 is preferred.

 Still other stabilizers such as sugars, such as dextran, hydroxyshetti

Substitute Sir G- One or more substances selected from polysaccharides such as starch, disaccharides such as sucrose, maltose and lactose, and monosaccharides such as glucose, fructose, mannose and galactose can also be added.

 Regarding the above-mentioned dextran and hydroxyxechinole starch, commercially available ones can be used, but in order to clinically apply the present composition, those having a quality such that it can be used as a plasma substitute for parenteral administration are preferable. Dextran has an average molecular weight of 10,000 to 100,000, and more than 40,000 to 70,000, while hydroxyethyl starch has an average molecular weight of 10,000 to 200,000, and] 9 more than 20,000 to 60,000. 200,000 are advantageously used.

 When the above-mentioned saccharide is added, it is preferable that the aqueous solution be contained in an IFN-r aqueous solution in an amount of 1 ^ or more, preferably 3 to 50 ^.

 The composition may further contain the above-mentioned saccharide or (and) amino acids as an osmotic pressure adjusting agent, but the addition of an inorganic salt such as sodium chloride may adversely affect the quality of the composition. Is inferior, so it is preferable. The above-mentioned preferable osmotic pressure adjusting agent may be added to the composition in advance, or may be added to a solvent for re-dissolving the freeze-dried product.

The frozen and freeze-dried human FN- _r composition of the present invention can be produced, for example, by the following method.

Substantially exist inorganic salts torquecontrol preparative IFN- rlxl O ² ~: LX to 1 0 ⁷ I-containing aqueous solution, the Amino acid l ^ / or more, preferably ¾ so that the 5-5 0 Roh) ^ concentration Add to The above-mentioned amino acid can be added to the aqueous solution containing IFN- _r during the production process. When an aqueous solution of IFN-r also containing this amino acid is used, it is necessary or necessary. Amino acids can be added up to the concentration of the amino acids and subjected to the following step. Change Further, the above-mentioned saccharides and the like can be added together.

The aqueous IF ¹ N-r solution may contain a reducing sulfur compound of at least 0.1 ^, preferably 0.5 to 1) ^ and a trace amount of a surfactant, and Like agents, these can be added fresh.

 The frozen human IFN-r composition of the present invention can be obtained, for example, by freezing the above aqueous solution at a temperature of usually 180 ° to −30! ) Can be manufactured. Preferably, the frozen composition is stored at 180 ° C to 110 ° C.

 The freeze-dried human IFN-r composition of the present invention can be obtained, for example, by drying the above-mentioned frozen composition under reduced pressure or by thawing the above-mentioned aqueous solution or the above-mentioned frozen composition! The resulting aqueous solution can be subdivided as desired], frozen in the same manner as described above, and dried under reduced pressure by a conventional method.

 When producing the freeze-dried human IFN-r composition of the present invention as an injectable preparation, the aqueous solution of the composition or its component is purified by sterilization and filtration before subdivision, and sterilized. It is preferable to dispense into a vial or the like by an operation and then subject to the freeze-drying treatment.

 The frozen or freeze-dried human IFN-r composition of the present invention has very little decrease in F-r activity and quality during the freeze-freezing or freeze-drying operation and storage, and becomes turbid when re-dissolved! ) Is useful because it does not occur. Also, the lyophilized composition was obtained as a stabilized human IFN-r powder! In particular, it can be advantageously used as a parenteral preparation.

When the freeze-dried human IFN-r composition of the present invention is used as a preparation for injection, the lyophilized composition is usually used in a vial, i.e., distilled water for injection or an injection solution of sugar, etc. Dissolve and use the solution within the physiologically acceptable range of osmotic pressure. It can be used as a dosage form for ophthalmic, otic and nasal administration using appropriate carriers, excipients and diluents. The frozen or freeze-dried human IFN-II composition of the present invention has low toxicity, and can be used for the same purpose as known human IFN-r and in the same manner.

 In the present specification, the international unit (I U) was determined as the activity of IFN-r (antiviral activity) as follows.

The international standard IFN- _r whose unit (unit) was determined and the target data were measured using the cytopathic effect inhibition test of Sindbis Winores (S indbi Virus) against FL cell line derived from human amniotic membrane, and the ratio was used to determine the power. The value was calculated.

 The amount of protein in the solution was determined by calculating E: 280 nm = 1.0 as 1 ^.

 BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 shows the amino acid sequence of IFN-r contained in the aqueous solution of rIFN-r disclosed in Reference Example.

 BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited thereto.

 As for I?-Used in the examples, a high-concentration aqueous solution of Hie-FN-r containing an inorganic salt and containing substantially an inorganic salt produced by the method described in Reference Example 1 was used unless otherwise noted. The chicken FN-r has the amino acid sequence shown in FIG.

The transformant Escherichia coli 2 94 p HIT trp 2101 disclosed in Reference Example 3 was obtained from the Ministry of International Trade and Industry of Deposit No. (FRI) and deposited under accession number FERMP-750. Difference _v Example 1

Eradication泸過to Gunoretachio down (reduced) obtained by ^{3 ^ 2. 4 1 0 6 I} ^ J ni of human IFN comprising - were sterilized泸過containing r aqueous 1 mi glycine 1 5 ^ 0.5 W of the aqueous solution was added, and lyophilized in a vial boat. The freeze-dried product was redissolved in distilled water for injection 1 and the solution was visually observed and the IFN-II titer was measured. The results are shown in Table 1.

Example 2

 Example 1 was carried out in the same manner as in Example 1 except that hydroxyzinole starch (average molecular weight: 200,000) 37.5 ^ was added together with glycine 15. The results are shown in Table 1.

Example 3

 Example 1 was carried out in the same manner as in Example 1 except that glycine was increased to 30 ^ and sodium glutamate (7.5 W) was further added. The results are shown in Table 1.

Example 4

Gunoretachion obtained by spent decontamination reactor (reduced) 3 ^ 3. 7 X 1 0 6 I i ml of human IFN-r aqueous solution spent sterilization furnace containing glycine 3 0 ^ 0 containing. 5 and lyophilized in a vial. The freeze-dried product was redissolved in distilled water for injection, and the solution was visually observed and the IFN-r titer was measured. .

 As a control, an aqueous solution of human IFN-r containing gnoretathione (reduced form) 3 ^ obtained through a sterilization oven without glycine was freeze-dried in a vial bottle, and the power of IFN-r was similarly determined. The titer was measured. The results are shown in Table 1.

Replacement Table 1

 *: Residual potency of lyophilized product relative to solution before lyophilization Example 5

Eradication泸過was obtained Darutachion (reduced) 3 ^ 4.6 XI 0 ⁶ E of human IFN-r aqueous 1 v glycine 3 0 ^ containing sterilization泸過was 7 solution 0.5 containing added After cryopreservation at 130 ° C for one week, the IFN-r titer was measured. The frozen product exhibited a titer of 7% with respect to the solution before freezing.

Example 6

R containing solution Darutachio emissions (reduced form) obtained by spent decontamination furnace 3 ^ and chloride na Application Benefits um ^{2.5 ^ 46 X 1 0 6 IUZ} l comprising <0 human - r IFN obtained in Reference Example 2 0.75 し た of the aqueous solution containing glycine 15 and passed through a sterilization oven was added to 0.25 of the aqueous IFN-r solution, and lyophilized in a vial. When the lyophilized product was redissolved in distilled water for injection 1, a small amount of fine insolubles was generated, and the IFΙίτ was titrated as it was.

 As a result, the titer of the human IF I-r solution before freeze-drying was 98%.

Example 7

2.6 X 1 0 ⁶ IU presumed dead, switch comprising a sterilization ^ spent which was obtained Darutachion (reduced) 3 ^ l Zm human IFN-r aqueous solution 0.5 was added to a sterilized filtered aqueous solution 0.25 containing glycine 1 o and hydroxyethyl starch (average molecular weight 40,000) 15 ^ and freeze-dried in a vial. . The lyophilized product was redissolved in distilled water for injection 0.5 to confirm that the solution was clear, and IFN- _r

A titration of 5 was performed.

 As a result, the titer of the human IFN-r solution before freeze-drying was 94%. In addition, the residual ratio to the titer at the start of storage after storage at 40 ° for one month was 100%, which was stable.

 Example 8

2.6 X 1 0 ⁶ containing 10 eradication泸過to give the glutathione (reduced form) 3 ^

 To an aqueous solution of human IFN-r of I ^ / nl 0.5 was added 0.25 of a sterilized filtered aqueous solution containing 15- ^ sodium gnooletamate and 15 W of hydroxyethynole starch (average molecular weight 40,000). Lyophilization was performed in vials. The freeze-dried product was redissolved in distilled water for injection 0.5 to confirm that the solution was clear, and the FN-r titer was measured.

 As a result, the titer of the human IFN-r solution before freeze-drying was 102%. The residual ratio to the titer at the start of storage after storage at 40 ° for one month was stable at 106%.

The same results as in the above example can be obtained by using the high-concentration rFN- _r water 20 solution substantially free of inorganic salts obtained in Reference Examples 3 to 5.

 Reference Example 1 Production of high-concentration rIFH-r aqueous solution containing substantially no inorganic salts

 I

(I) A strain RRl (pR248cIts, 25 pRC23I / IF1-900) having a working human IFN-r gene was prepared according to the description in Example 8 of EPC 0 08 9 6 7 9 Cultured frozen cells were replaced with 100 M by 7 M Add 300 mM 100 mM Tris-HCl buffer (ρΗ7.0) containing guanidine hydrochloride and 2 mM phenylmethinolesulfonyl phenololeide, stir at 4 ° C for 1 hour, and centrifuge (17,000 rp 0 min) to obtain a clear supernatant. This supernatant was diluted with 1337 mM sodium chloride, .27.

mM calcium chloride, 8 mM sodium phosphate and 0.147 mM phosphate

 The mixture was diluted 70-fold with a buffer solution containing potassium (hereinafter abbreviated as PBS), and the resulting precipitate was removed by a Sharples centrifuge (10, OO rpm). Then, the obtained supernatant solution 220 was subjected to Pellicon (Millipore Corporation).

·, Fractional molecular weight: 10,000) and concentrated to 15 Add this concentrate to 4

The mixture was allowed to stand at ° C overnight, and the resulting precipitate was further removed by a Sharpless centrifuge. 5 × 30 antibody column [AD

 (M 0 ■ r 2-11.1);

 12)) at a flow rate of 1,000 i, hour,

 Contains 2,500 ^, 1 sodium chloride and 0.1% Tween 20

 5,000 ^ ,? of 10 mM phosphate buffer (pH 7.0) 33 After passing through each of the 2,500 µl washings of 20 mM phosphate buffer (pH 7.0) containing 2,500 and 0.5 M guanidine hydrochloride sequentially through the antibody column, 2 M hydrochloric acid was added. The column was eluted with 20 mM phosphate buffer (pH 7.0) containing guanidine, and the eluted fraction 50 having anti-viral activity was collected.

 (S) To the eluted fraction 42 obtained by the method of Reference Example 1 (I), 10 mM of reduced daltatin was added. Prepare 4 20 of this human IFN-r aqueous solution in advance.

 I mM ethylenediaminetetraacetate, 150 m sodium chloride, 10

A column (9 × 10 Oc) of Sephacryl Nole S—200 (manufactured by Falmecia) equilibrated with 25 mM acetate buffer (pH 6.0) containing 2 mM glutidine hydrochloride and 2 M guanidine hydrochloride. ), And exchange with the same buffer solution., -W1PO,. The monomer eluted fraction 450 was collected. By the present operation]? To give a specific activity 3.4 X 1 0 ⁶ I UZ ^ protein of IFN- r (0.4 1 0 m / ml).

(H) Reference Example 1 Human IFN-r (monomer) eluted fraction obtained in (H) 450 i10 reduced glutathione, 150 mM sodium chloride, 0.5 M guanidine hydrochloride and 0.01% 3,240 t of a diluted solution of 25 mM acetate buffer (pH 6.0) containing Tween 20 was added and mixed to prepare a low-concentration solution having a protein content of 0.05. This solution was preliminarily equilibrated with 25 mM acetate buffer (pH 6.0) containing 10 mM reduced daltathione, 150 mM sodium chloride, and 0.01% zinc 20 (Sephadex). Elution fraction of human IFN-r, loaded on a column of G-25 (14 x 100, gel filtration with the same buffer, and guanidine hydrochloride) 3,180 (155.8 ^) The amount of the protein in the solution was 0.049.The recovery rate of the protein was 84.4% and the specific activity was 3.5 X 10 ^s I UZ ^ protein. After aging for 48 hours at 4 ° C, the solution was concentrated to 159 by ultrafiltration using Diaflow PM-10, 43 ram ø (Ultimate membrane manufactured by Amicon). The concentrate was clear and had a protein content of 0.92 ^ /) ^. The protein recovery was 93.9% (146.3 ^). All human IFN- r of the specific activity is ^{6.8 1 0 6 IU / »¾} ? Data down white Der ivy

(W) An aqueous solution (protein content: 0.952 / nl) containing a high concentration of human IFN-r obtained by the method described in the above (3) was preliminarily containing 10 mM reduced daltathione. 25 Loaded on a Sephadex G-25 column (5.0 × 50.Ocffl) equilibrated with 25 mM acetate buffer (pH 6.0), developed with the same buffer, and had a protein content of 0.589 9 ^ Substantially contains inorganic salts of Z ¾ ぃ (less than 10 ppm) clear ¾ IFN-r solution 5 was obtained. Replacement '' The specific activity of this IFN-r was 3.7 x 10 ⁶ ΙΠ / ^-protein.

Reference Example 2 Production of High Concentration r I F Ν -r Aqueous Solution

 Reference Example 1 3 of an aqueous solution (protein content; 0.952 / ml) containing human IFN-r in a high concentration obtained by the method of (Π) was previously added to 10 mM reduced gnoretathion and 40 mM 25 mM acetate buffer containing sodium chloride

Sephadex G-25 column (equilibrated with pH 6.0) (Loaded to 5.0 x 50.0, developed with the same buffer, contains 0.04 M sodium chloride with a tin white content of 0.658 Z) A clear IFN.-r solution of 52 w was obtained.

The specific activity of this IFN-r was 4.6 × 10 ⁶ IU ^ · protein.

Reference Example 3 Production of High Concentration r I F Ν -r Aqueous Solution Containing Virtually Inorganic Salt

 n

 (I) Transformation described in Reference Example 2 of EPC 0 1 0 3 988 Publication. Culture of recombinant Escherichia coli 294 / pHIT trp 2101, 7 M guanidine hydrochloride to frozen cells 1 Add 0.05 M boric acid buffer (pH 7.2) to the solution, stir at 4 ° C for 1 hour, and centrifuge (17,000 rpm / 30 minutes) 700 W. This extract was added to a buffer solution consisting of 0.137 M sodium chloride, 2.7 mM sodium chloride, 8 mM sodium phosphate and 1.47 mM sodium phosphate (lower PB). S (abbreviated as S). Next, 0.4 silica gel (Separation Industries, Ltd.) was added to the diluted solution, stirred at 4 ° C for 45 minutes, allowed to stand for 15 minutes, and the upper solution was discarded by the tilt method. This sieve was thoroughly washed with a 0.05 M phosphate buffer (pH 7.2) containing 1 M sodium chloride, and then packed into a column (11 × 11 ^). Next, elution was performed with a 0.01 M borate buffer (pH 8.0) containing 0.5 M tetramethylammonium chloride, and the eluate 20 was subjected to a 5 × 8 antibody ram [Ab

 -

^ ^. レ ^ O ^ MP! _ After washing with 750 EBS, elution was performed with 0.02 M phosphate buffer (pH 7.0) containing 2 M guanidine hydrochloride. Abbreviation) Fraction 183 having activity was collected. To this eluate was added 0.01 M amount of reduced glutathione (562) to obtain an aqueous solution containing 2.1 × 10 ⁶ I ^ / 'protein rIFN-rl72 ^.

 (H) Reference Example 3 rIFN obtained in (I) —183 »of the aqueous solution was prepared in advance using 1 mM ethylenediaminetetraacetate, 0.15 M sodium chloride, 0.01 M reduced dartathion and 2 Loaded onto a Sephacrel S — 200 (Pharmacia) column ('9779 ^) equilibrated with 25 mM acetate buffer (pH 6.0) containing M guanidine hydrochloride and developed with the same buffer Then, a monomer-eluting fraction 433 was collected. The fraction obtained in this manner converged to the monomer by slab electrophoresis of sodium dodecyl sulfate (hereinafter abbreviated as SDS-PAGE). Specific activity 3.0 X 10 IU / ' "An aqueous solution containing 142 ^ IFN-r was obtained.

(H) Add 163.4 of 25 mM acetate buffer (pH 6.0) containing 0.01 M reduced daltathione and 0.5 M guanidine hydrochloride to 36.6 (12.0) of the aqueous solution containing rIFN-r obtained in (H) above. Then, a 0.06 concentration solution was prepared at 200 ° C. This solution was loaded onto a Sephadex G — 25 (Pharmacia) column (5 x 60 cm) previously equilibrated with 25 mM acetate buffer (pH 6.0) containing 0.01 M reduced gnorethatione. Then, elution was performed with the same buffer to obtain rIFN-r fraction 220. The FN-γ concentration of this solution was 0.047 / d. The solution was then aged for 2 days at 4 and then concentrated to 14 by the ultrafiltration method of Diaflo PM-10 membrane (Amicon's ultra furnace membrane), with a protein content of 0.711 ^ Substitute in real A clear rIFN-r solution that does not contain inorganic salts (less than 10 ppm) was obtained. The thus obtained high concentration rIFN-r converged on the monomer by SDS-PAGE. The specific activity of r I Ϊ ¹ N—r was 3.6 10 ⁶ KW-protein.

 Example Production of high concentration r I F N -r aqueous solution containing substantially no inorganic salts H

 Reference Example 1 One of 25 mM acetate buffer (pH 6.0) containing 0.01 M reduced glutathione and 0.5 M guanidine hydrochloride in 35 (11.48) of the r IPN-r aqueous solution obtained in (I) The 0.057-NOW concentration solution 200 prepared by adding 65 was loaded onto a Sephadex & 25 column (5 x 60 cm) previously equilibrated with 0.01 M reduced glutathione solution (pH 6.0). It was developed with a 0.01 M reduced glutathione solution (pH 6.0) to obtain an eluted fraction 23 of rIFN-r. The rIFN-r concentration of this solution was 0.046. The solution is then aged at 4 ° C for 1 day, then concentrated to 10 by ultrafiltration using a Diff-Mouth PM-10 membrane.

1.08 ^^^ gave a clear FN-r aqueous solution substantially free of inorganic salts (less than 10 ppm) and acetate buffer. The thus obtained rl FN-r converged on the monomer by SDS-PAGE. The specific activity of this rFN-r was 3.6 × 10 ⁶ · tan.

Example 5 Production of a high concentration rIFN-r aqueous solution containing substantially inorganic salts

 W

35 mi of rIFN-r obtained in Reference Example 101) (16.5) of 25 mM acetic acid buffer solution (pH 6.0) containing reduced daltathione and 0.5% guanidine hydrochloride in 11.48¾¾ VI was added to prepare a 0.057 ^; ^ concentration solution of 200. This solution was previously replaced with 0.267 M glycine and 0.01 M reduction. The column was loaded on a Sephadex G-125 column (5 x 60) equilibrated with a solution (pH 6.0) containing glutamic acid, developed with the same solution, and the eluted fraction of rIFN-γ 2 202 ^ I got it. The rIFN-r concentration of this solution was 0.046. This solution is then aged for 2 days at 4 and then concentrated to 9.4 by ultrafiltration on a Diaflow PM-10 membrane, with a tan white content of 1.07 / substantially inorganic salt (less than 10 ppm). ) And clear FN-r solution without acetate buffer. The r _IFN-r is converged to the monomer in SDS-PAGE, the specific activity thereof 3.6 2 1 0 ⁶ I UZ - was data down white.

 Industrial applicability

Substantially absent inorganic salts of the present invention, human I 5 ¹ N of amino acid was sintered or lyophilized freeze under conditions that coexist - 7 "compositions, the frozen or freeze-drying operation and during storage In this case, the decrease in FN-r activity is extremely small, and no turbidity 9 is produced upon re-dissolving, so that it can be advantageously used as a pharmaceutical or the like.

Replacement

Claims

The scope of the claims  1. A human r-type interferon composition frozen or lyophilized under conditions substantially free of inorganic salts and amino acids.  2. The composition according to claim 1, wherein the amino acid is a monoamino aliphatic amino acid. OMPI WH> 0 ~ * : ^ 捭 ぇ,