WO1986006080A1 - gamma-INTERFERON COMPOSITION - Google Patents

Abstract

Human gamma-interferon composition which contains substantially no inorganic salts and which is frozen or lyophilized in the presence of an amino acid. A substantially inorganic salt-free aqueous solution of human gamma-interferon can be prepared by using an inorganic salt-free buffer solution as a buffer solution in the final chromatographic procedure or by removing inorganic salts from a purified aqueous solution. When the solution is frozen or lyophilized after adding thereto an amino acid, the activity of gamma-interferon is scarcely reduced during the procedure, and the resulting composition has good storage stability and, since no turbidity is produced upon re-dissolution, it can be advantageously used as antiviral agent, antineoplastic agent, etc.

Description

 Specification

 A type interferon composition

 Technical field

 The present invention relates to a heat-type interferon composition.

 Background technology

 The human interface is currently classified into three types: type, type 3 and type a. α- and ^ -type interferons 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, type 7 interferon (sometimes abbreviated as IF-pair) is extremely unstable and easily loses its activity or freezes during storage, freezing or lyophilization of aqueous solutions. There are problems such as rejection of the dried product after re-dissolving, making it extremely difficult to obtain a stable composition worthy of clinical use. Therefore, it has a remarkably strong antiviral and antitumor activity among interferons [Salvin et al., Journal of National Cancer Institute, Vol. 55, p. 1233 (1975)]. It is a big bob for clinical application of IFN-—, which is the most promising drug.

 By the way, the drug substance of interferon to be used in pharmaceutical preparations is usually obtained as a high-purity product from a crude interface through a separation and purification process using various types of chromatography and the like. In the separation and 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 interferon. It was thought that it would also work advantageously as a stabilizing agent when formulated.

For example, it has been disclosed that ^ -type interferon having no sugar chain can be stabilized by adding an inorganic salt (Japanese Patent Laid-Open No. No. 19—25364).

 Under such technical background, the present inventors surprisingly formulate a pharmaceutical preparation using an aqueous IFN-a solution in which the concentration of an inorganic salt is reduced. With respect to δ in the above operation, it was found that the IFN-α activity was further reduced, and that a stable IFN-α composition was obtained in which a solution obtained by re-dissolving the obtained composition did not show any swelling. The present inventors completed further research and completed the present invention.

 Disclosure of the invention

 The present invention provides a human-type interferon composition which is substantially free of inorganic salts and free from amino acids or freeze-dried or freeze-dried under cows.

 More specifically, rlFN-7 includes a polypeptide consisting of 146 amino acids and various fragments of the polypeptide exemplified in FIG. Various fragments include, for example, N-terminal-deleted splices in which four or less amino acids at the N-terminal part of the polypeptide are deleted; 31 Examples include C-terminal missing pieces cut at the site after the amino acid residue. Furthermore, the above rIFN-r also includes those in which the cysteine residue of the above polypeptide is substituted with serine or threonine.

0 Among the above various fragments, the N-terminal part of the polypeptide consisting of 146 amino acids shown in Fig. 1 lacking four or less amino acids at the N-terminal part of the polypeptide It is preferable that the C-terminal portion of the species or the N-terminal-less fragment is truncated.

 In particular, as the human IFN-a of the present invention, 1 4 6 δ 1 shown in FIG.

Of amino acid or Cys of the polypeptide 2 3] _ 2 3

 Tyr-Cys-missing spices [Des (Cys-Tyr-Cys) IF-r] are preferred.

 Also, an aqueous solution containing human IFN-7 at a high concentration obtained by genetic recombination technology is advantageously used.

Human IF - deactivation of § is 1 is preferably X 1 is 0 ⁵ ~ 1 XI 0 ⁷ international units Zmg (I UZmg), as the IFN- § ^{aqueous, IX 1 0 2 ~ 1 X} 1 0 7 Those having an activity of IU / ml, especially 1 ^× 10 ⁺ to 1 ^× 10 ⁷ IU / l are preferred.

 As the above IFN-a aqueous solution, one containing substantially no inorganic salt is used. Here, the concentration of the inorganic salt may be Q.1M or less, preferably 0.05M or less, particularly preferably 1 mM or less. . The inorganic ion intensity may be an ion intensity calculated from inorganic ions of 0.1 or less, preferably 0.05 or less, particularly preferably 0.01 or less. Further, in the freeze-dried composition, the inorganic salt is preferably at most 30%, more preferably at most 15%, particularly preferably at most 0.3%, based on the total weight thereof.

 The aqueous solution of IFN-a substantially free of inorganic salts can be obtained, for example, by using a buffer solution containing no inorganic salts as a buffer used in the purification step of IFN-y, particularly in the final step of chromatography, or by using purified IFN-y. —A Can be produced by removing inorganic salts from an aqueous solution.

As the amino acid, monoamino aliphatic amino acids such as daricin, α-alanine, 3-alanine, leucine, glutamic acid, and aspartic acid 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, and the amino acids used are those that can be used in Osaka. For clinical application of the product, it is preferable that it be of a quality that can be used for parenteral administration. ·

 It is preferable that the total amount of the acid and the acid is 1 mg or more, preferably 5 to 50 mg, per 1 ml of the IFN-7 aqueous solution.

 In the composition of the present invention, a reducing sulfur compound may be allowed to coexist when IFNa has a cysteine residue. Examples of the reducing sulfur compounds include glutathione (reduced form), thioctic acid, thiodiglycol, thioethanolamine, monothioglycerol, dithiothreitol, and thioalkanoic acid having 1 to 7 carbon atoms. Glutathione (reduced form) is particularly preferred. When a reducing sulfur compound is allowed to coexist, the reducing sulfur compound is preferably 0.1 mg or more, particularly preferably 0.5 to 1 Omg per IFN-'/ l ml aqueous solution.

 Still other stabilizers may include human serum albumin (HSA) or (and) glucose. The HS A may be of any type, but for the clinical application of the present composition, it is preferably of such a quality as to be used for parenteral administration. For example, fractionated and purified from healthy human plasma by the C'ohn ethanol fractionation method 6 [Journal of American Chemical Society, Vol. 68, 4: 59-475] (1946)] is used. Further, HSA may contain acetyltriptosodium sodium or sodium caprylate as a stabilizer.

 HS A is more than 1 mg / ml of aqueous solution of IFN-

It is preferable to contain 2 mg to 20 mg.

In the case where the composition of the present invention contains a HS A is in solution to adjust to indicate pH 4.0 to 5.0 or 7.5 to ^8.5: Rukoto are preferred. More specifically, it is preferable to adjust the pH to 4.0 to 5.0 when the amino acids are neutral amino acids, and to adjust the pH to 7.5 to 8.5 when the amino acids are acidic amino acids. ' The saccharides are selected from, for example, polysaccharides such as dextran and hydroxyxetyl starch, disaccharides such as sucrose, maltose and lactose and monosaccharides such as glucose, fructose, mannose and galactose. One or more substances may be mentioned.

 Regarding the above-mentioned dextran and hydroxyxetil starch, commercially available ones can be used. However, in order to clinically apply the present composition, those having a quality enough to be used for parenteral administration as a plasma substitute are preferable. Dextran has an average molecular weight of 10,000 to 100,000, especially 40,000 to 70,000. Are advantageously used.

 When the above-mentioned saccharide is added, it is preferable that the saccharide be contained in an amount of 1 iig or more, preferably 3 mg to 50 mg, per 1 ml of the aqueous solution of IFΝ- ァ.

 The present composition may further contain the saccharide or (and) amino acid as an osmotic pressure regulator, but the addition of an inorganic salt such as sodium chloride is warped. It is not preferable because it deteriorates the quality of the composition. 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 IFN-A composition of the present invention can be produced, for example, by the following method.

Substantially human IFN no inorganic salts - § ^{1 X 1 0 2 ~ 1 X} 1 0 7

Amino acids are added to the aqueous solution containing I / ml to a concentration of 1 mg / ml or more, preferably 5 to 50 mg / ml. The above-mentioned amino acid can be added to the aqueous solution containing IFN-α during the production process. When an aqueous solution of IFN-α also containing this amino acid is used, the aqueous solution of IFN-α can be used as it is or as necessary. Amino acid can be added up to the concentration of amino acid and subjected to the following steps. Further, the above-mentioned HSA and sugars can be added together.

 The aqueous IFN-a solution may contain a reducing sulfur compound or a trace amount of a surfactant of Q.lmg / ml or more, preferably Q.5 to 10 mg / ml, and like the above-mentioned stabilizer, These can be newly added.

 If desired, adjust the pH by adding an aqueous solution of mineral acid (hydrochloric acid, sulfuric acid, etc.) or (and) inorganic base (sodium hydroxide, sodium carbonate, etc.).

 The frozen human IFN-A composition of the present invention can be produced, for example, by freezing the above aqueous solution usually at 180 to 130 ° C. Preferably, the frozen composition is stored at 180 ° to 110 ° C.

 The freeze-dried human IFN7 composition of the present invention may be obtained by, for example, drying the above-mentioned frozen composition under reduced pressure by an ordinary method, or subdividing the above-mentioned aqueous solution or an aqueous solution obtained by thawing the above-mentioned frozen composition, if desired, and freezing as described above. After that, it can be manufactured by drying under reduced pressure by a conventional method.

 When preparing the freeze-dried human IFN-α composition of the present invention as an injectable preparation, the aqueous solution of the composition or its components (each of which is purified by sterilization filtration or the like) before the subdivision is subjected to aseptic operation. And then freeze-drying is preferably performed.

 The frozen or freeze-dried human IFN-α composition of the present invention has a very small decrease in IF-α activity and quality during the freeze-freezing or freeze-drying operation and during storage, and does not cause shuffling during re-thawing. Useful. The freeze-dried composition is obtained as a stabilized human IFN-a powder and can be advantageously used particularly as a parenteral administration preparation. In this case, the composition to which HSA is further added has little adhesion to the vessel wall and can be advantageously used.

The freeze-dried human IFN-α composition of the present invention is used as an injection preparation. In normal use, dissolve the lyophilized composition in 1 to 10 O ml of distilled water for injection or budou sugar injection solution per vial for normal use, and the osmotic pressure of the solution is within the physiologically acceptable range. Used in. It can also be used as ophthalmic, otic and nasal dosage forms using appropriate carriers, excipients and diluents.

 The frozen or freeze-dried human IFN-'/ composition of the present invention has low toxicity and can be used for the same purpose as that of the known human IFN-α by the same usage.

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

 The international standard IFN-α with a defined unit (unit: soto) and the target material were compared with the Sindbis virus (S indbis) against FL cell line derived from human amniotic membrane.

Virus), and the titer was calculated from the ratio.

 The amount of protein in the solution was calculated by assuming E: 280 nm = 1.0 as 1 mg.

 Brief description of the drawings

 FIG. 1 shows an example of the amino acid sequence of rIFN—α consisting of 146 amino acids. FIG. 2 shows a construction diagram of the plasmid PLC2 disclosed in Reference Example 6 (i).

 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.

Unless otherwise noted, the IFN-α used in the Examples was a high-concentration human rIF-α aqueous solution substantially free of inorganic salts produced by the method described in Reference Example 1. The human rIFX-α has the amino acid sequence shown in FIG. The transformant Escherichia coli 294 / HI Ttr 2101 disclosed in Reference Example 3 was obtained from March 19, 1980, on March 19, 1980 at the Research Institute of Microbial Industry (FRI) Deposit No. FE RM P — 7550.

 In the production of the compositions described in Examples 1 to 8, the pH was not actively adjusted, but the pH at the time of reconstitution with distilled water for injection was in the range of pH 5.5 to 7.

Example 1

Glutathione obtained by Joran filtered (reduced form) solution was sterile filtration 3mg to 2.4X 1 0 of ⁶ IU / ml human IFN-7 aqueous solution 1 ml containing containing glycine 1 5 mg 0.5 ml In addition, lyophilization was performed in vials. The lyophilized product was redissolved in 1 ml of distilled water for injection, and the solution was visually observed and IFN-7 was titrated. The results are shown in Table 1. · Example 2

 Example 1 was carried out in the same manner as in Example 1 except that 157.5 mg of glycine was mixed with 37.5 mg of hydroxyxethyl starch (average molecular weight: 200,000). The results are shown in Table 1.

Example 3

 Example 1 was carried out in the same manner as in Example 1 except that the amount of glycine was increased to 3 Omg and 7.5 g of sodium glutamate was further added. The results are shown in Table 1.

Example 4

Joran filtered obtained was glutathione (reduced form) containing 3mg 3.7x 1 0 ⁶ IU / ml of human IFN- § aqueous 1 mi solution was sterile filtration containing glycine 3 0 mg to 0.5ml In addition, lyophilization was performed in vials. The freeze-dried product is redissolved in 1 ml of distilled water for injection, and the solution is visually observed and IFN-α Was measured.

 Without adding glycine as an elephant, iml of an aqueous solution of human IFN-α containing 3 mg of glutathione (reduced form) obtained by sterilization filtration was freeze-dried in a vial, and the IFN- Was measured. The results are shown in Table 1.

 Table 1

 Example of application

 1 Clear 1 0 0 ¾

 2 Clear 9 4%

 3 Clear 104%

 4 Clear 9 8%

 4 (Control) Clear 8 6%

 Residual potency of lyophilized product relative to solution before lyophilization

Example 5

The aqueous solution 0.5ml was sterile filtration containing human IFN- § aqueous 1 mU this Dali Shin 3 0 mg of 4.6X 1 0 ⁸ IU / ml including sterile filtration obtained was glutathione (reduced form) 3 mg In addition, −3 ° C. for one week. After cryopreservation, IFN-α titer was measured. The frozen product exhibited a titer of 97% of the solution before freezing.

Example 6

R IFN obtained in Reference Example 2 - y containing solution Gurutachio emissions (reduced form) obtained by sterilization filtration 3 mg / [pi and humans in 4.6X 1 0 ⁶ IU / nU containing chloride Natoriumu 2.5mg Bruno ml 0.75 ml of a sterile-filtered aqueous solution containing 15 mg of daricin was added to 0.25 ml of the IFN-aqueous solution, and lyophilized in a vial. When the freeze-dried product was redissolved in 1 mL of distilled water for injection, a small amount of fine insoluble matter was generated, and the IFX-I was titrated as it was. 1 As a result, at 98% of the titer of the human IFN-

' there were.

 Example 7

Darutachion obtained by sterile filtration (reduced) 3 mg / ml of 2.6x 1 0 ⁶ 5 I UZml containing human IFN- § aqueous 0.5mU this glycine 1 0 rag and inhibit mud Kishechiru starch (average molecular weight 4 0.25 ml of a sterile-filtered aqueous solution containing 15 mg was added and freeze-dried in a vial. The freeze-dried product was redissolved in 0.5 ml of distilled water for injection to confirm that the solution was clear, and the titer of IFN-α was measured.

 As a result, it was 94% of the titer of the human IFN-a solution before freeze-drying.

• there were. In addition, the residual ratio with respect to the titer at the start of storage after storage at 40 C for 1 month was 100%, which was stable.

 Example 8

Sterile filtration to give glutathione (reduced) 3 2.6x 1 comprising mg ml 0 ⁸ 5 I glutarate the human IFN-7 aqueous solution 0.5ml of UZml Mi Nsan'na Application Benefits um 1 5 mg and human Dorokishechiru starch (Average molecular weight: 40,000) Disinfection containing 15 mg 0.25 ml of a filtered aqueous solution was added and freeze-dried in a vial. The freeze-dried product is redissolved in 0.5 ml of distilled water for injection to confirm that the solution is clear.

IF-r titrations were performed.

0 As a result, the titer of the human IFa solution before freeze-drying was 102%. In addition, the residual ratio to the titer at the start of storage after storage at 40 ° C for one month was stable at 106%.

 Example 9 '

Darutachion human in 3.8X 1 0 ⁸ I UZnil containing (reduced) 3 mg / ml I Fδ N - § solution 0.16 ml, containing HSA 5 mg / mi and glycine 2 3 mgZml, pH4 with HC 1. 1 ml each of the sterilized and filtered aqueous solution prepared in 5 The mixture was dispensed into a vial and freeze-dried. The lyophilized product was redissolved in 1 ml of distilled water for injection, and the solution was visually observed, pH was measured, and IFN-7 was titrated. The results are shown in Table 2.

Example 10

 Example 9 was carried out in the same manner as in Example 9 except that the amount of HS A was increased to 10 mg / ml. The results are shown in Table 2.

Example 11

 Example 10 was carried out in the same manner as in Example 9, except that HS A was reduced to 20 mg / ml. The results are shown in Table 2.

Example 12 4 5465

 Example 9 was carried out in the same manner as in Example 9 except that hydroxyxethyl starch (average molecular weight: 40,000) was further added to 5 mgZml. The results are shown in Table 2. ―

Example 13

 Example 10 was carried out in the same manner as in Example 10 except that sodium glutamate was added instead of glycine to 27 mg / ml to adjust the pH to 7.8 with a 0.1 M NaOH aqueous solution. The results are shown in Table 2. Table 2

 Example Welded PH; Residual titer *

 9 Clear 1 0 7%

 1 0 Clear 1 0 5%

 1 1 Clear light -9 8%

 1 2 Clear 1 0 9%

9 5 % 1 3 Akira Akira

9 5%

Residual potency of lyophilized product relative to solution before lyophilization -12-

1 Even when the high-concentration rIFN-aqueous solution substantially free of inorganic salts obtained in Reference Examples 3 to 5 is used, the same effect as in the above-described example can be obtained.

 Example 14

2.5X 1 was obtained by the method described in 1 2 Example ⁶ 0 6 IU / ml of des (Cys- Tyr o

 Three

 1 Cys) IF-r aqueous solution 0.25n, containing 1mg / ml of HSA- 5mg / ml and 23mg / ml of glycine and adjusted to pH 4.5 with Q.1N HC1 Dispensed into bottles and lyophilized. The freeze-dried product was redissolved in 1 ml of distilled water for injection. The solution was clear and had a pH of 4.5. Ma

The residual ratio to the potency of the aqueous solution before freeze-drying was 96%.

 Example 15-

2.5X 1 was obtained by the method described in 1 2 Example ⁶ 0 6 IU / ml of des (Cys- Tyr

3 '

 -Cys) IF-aqueous solution · 0.25πι1, sterilized and filtered aqueous solution adjusted to contain 15 mg / ml of hydroxyxethyl starch (average molecular weight of 40,000) and 23 mg / ml of daricin ml was also dispensed into vials and lyophilized. When redissolved in the same manner as in Example 14, the solution was clear and had a pH of 6.5. The residual ratio to the potency of the aqueous solution before freeze-drying was 101%.

 Reference Example 1 Production of a high-concentration r l F N—a aqueous solution containing substantially no inorganic salts

(I) Strain RR1 (pRK248cIts, PRC231 / IFI-) having a working human IFN-a gene according to the description in Example 8 of EPC 0 896 979 The frozen cells obtained by culturing 900) contain 7 M guanidine hydrochloride and 2 mM phenylmethylsulfonyl fluoride.

100 Add Tris-HCl buffer (PH7.0) to 3QQ0na and shake for 4 hours for 1 hour. After stirring, the mixture was centrifuged (17,000 rpm / 30 minutes) to obtain a clear supernatant liquid. This supernatant was treated with a buffer solution (hereinafter abbreviated as PBS) consisting of 1337 sodium chloride, 27 mM potassium chloride, 8 mM disodium phosphate and 147 mM potassium phosphate. After dilution by a factor of 0, the resulting precipitate was removed by a sharpless centrifuge (10,000 rpm). Next, the obtained supernatant solution 220 β was concentrated to 15 β with Pellicon (Millipore, fractional molecular weight: 10,000). The concentrate was allowed to stand overnight at 4 ° C., and the resulting precipitate was further removed by a sharpless centrifuge. A 5 × 3 Octn antibody column [ΑΚΜο · α2—11.1); which was pre-filled with the supernatant, was applied to a flow rate of 1,00 Ond / After loading for 2.5 hours, add 2.5 ml of PBS, 5.0 ml of 10 mM phosphate buffer (PH7.0) containing 1 M sodium chloride and 0.1% Tween20. After successively passing 2.5 ml of Oml.PBS and 250 ml of 20 mM phosphate buffer (PH7.0) containing guanidine hydrochloride through the antibody column, The column was eluted with 20 mM phosphate buffer (PH 7.0) containing 2 M guanidine hydrochloride, and 500 ml of the eluted fraction having antiviral activity was collected.

(Ii) 10 mM reduced glutathione was added to 420 ml of the eluted fraction obtained by the method of Reference Example 1 (I). Preliminarily, 420 ml of this human IFN-aqueous solution was added to a 25 mM acetate buffer containing 1 mM ethylenediamine tetraacetate, 150 mM sodium chloride, 10 mM reduced glutathione, and 2 M guanidine hydrochloride. Was loaded onto a column (9 x 100 cm) of Sephacryl S-200 (Pharmacia) equilibrated with the same solution (PH6.0) and eluted with the same buffer. δ 0 ml was collected. To obtain a specific activity .4X 1 0 ⁶ IU / mg protein of IFN- 7 (Q.41Qmg / ml) by this operation.

(I) Reference Example 1 Add 50 mM reduced glutathione, 150 mi sodium chloride, 0.5 g sodium chloride to 450 ml of the eluted fraction of human IFN-monomer (monomer) obtained in (Π). Add and mix 3,240 ml of an acid buffer (PH6.0) diluted solution (25 mM) containing anidine and 0.01% tween 20 and a low concentration solution with a tan white content of 0.05 mg / ml. Was adjusted. This solution was preliminarily equilibrated with 25 mM acetate buffer (pH 6.0) containing 10 mM reduced glutathione, 150 mM sodium chloride and 0.01% Tween 20, pH G: 2 Load onto column 5 (14 x 100 cm), filter by gel with the same buffer, and elute fraction of human IFN-a from which guanidine hydrochloride has been removed 3,180 ml (155.8 mg) I got The protein content of this solution was 0.049 mg / ml. The protein recovery was 84.4%. Its specific activity was 3.5X 1 0 ⁶ I UZmg data down white. This solution was aged at 4 ° C. for 48 hours, and then concentrated to 159π by ultrafiltration using Diaflow PM-10, 43 (an ultrafiltration membrane manufactured by Amicon). The concentrate was clear and had a protein content of 0.92 mg Zml. The protein recovery rate was 93.9% (146.3 mg). Incidentally, the specific activity of human IFN- § is 6.8 X - Atsuta in 1 0 ⁶ I UZjng protein.

 (IV) An aqueous solution containing human IFN-y obtained at a high concentration obtained by the above method (ΠΙ)

(Separade's Sox G-25 column (38 ml of protein content; OJ mgZml) previously equilibrated with 25 mM acetate buffer (PH6.0) containing 1 OmM reduced daltathione ( 5.0 × 50. Ocm) and developed with the same buffer to obtain 57 ml of a clear IF 17 solution substantially free of inorganic salts (less than 1 Oppm) having a protein content of 0.589 mg / ml.

The specific activity of the IFN- § was 3.7X 1 0 ⁶ IU / mg · protein. Reference Example 2 Production of high concentration rl F-a aqueous solution

Reference Example 1 38 mi of an aqueous solution (protein content: 0.952 mg / ml) containing human IFN-7 at a high concentration obtained by the method of (m) was previously dissolved in 10 mM reduced glutathione and 4 OmM chloride. The column was loaded on a Sephadex G-25 column (5.0 X 50. Ocm) equilibrated with 25 sodium acid buffer (pH 6.0) containing sodium. The solution was developed with one buffer to obtain 52 ml of a clear IFN-α solution containing 0.04 M of sodium chloride having a protein content of 0.658 mg / ml.

The specific activity of the IFN- § was found to be 4.6X 1 0 ⁶ IU / mg · protein.

Reference Example 3 Production of a high-concentration rIF-r aqueous solution containing substantially no inorganic salts

 Π ―.

(I) Culture of the transformant Escherichia coli 2 94 / pH IT trp 2101, described in Reference Example 2 of EP C 0 i 0 3988, Publication of Reference Publication 1; Add 3.0 M O2 of 0.05M borate buffer (PH7.2) containing 7M guanidine hydrochloride to the mixture, stir at 4 ° C for 1 hour, and perform centrifugal separation (17,000 rpm / 3 0 Min) to obtain 2,700 ml of a clear extract. This extract was diluted 10-fold with a buffer solution consisting of sodium chloride, 2.7 mM potassium chloride, 8 mM sodium phosphate and 1.47 mM potassium phosphate (hereinafter abbreviated as PBS). Then, 0.4 kg of silica gel (Separation, Indus' Tries) was added to the diluted solution, stirred at 4 ° C for 45 minutes, allowed to stand for 5 minutes, and the upper solution was discarded by a gradient method. The silica gel was sufficiently washed with a phosphate buffer (PH 7.2) containing 1 M sodium chloride, and then packed in a column (11 × 11 cm). Then elute with 0.01M boric acid buffer (pH 8.0) containing tetramethylammonium chloride, and elute 2 (H is 5 x 8 cm antibody column [AKMo ♦ γ2-11.i); Out] load, 75 0 | 111? B. After washing with S, contain 2 M guanidine hydrochloride 0.02

The fraction was eluted with M phosphate solution (pH 7.0), and a fraction (183 ml) having antiviral (hereinafter abbreviated as AV) activity was collected. The 0.01M amount reduced glutathione in the eluate (5 6 2 rag) was added and specific activity ^{2. IX 1 0 6 IU / mg} · protein of r IF - to give a solution containing r n2mg.

 (Π) 183 ml of the rIFN-a-containing aqueous solution obtained in Reference Example 3 (I)

1 Ethylenediaminetetranate, sodium chloride, 0.01M reduced type 負荷 Load Sephacryl S-200 (Pharmacia) column (9 x 79 cm) equilibrated with 25 mM acetate buffer (PH'6.0) containing glutathione and 2 M guanidine hydrochloride. Develop with the same buffer and elute fraction of monomer

43.3 ml were collected. The fraction obtained in this way converged to the monomer by sub-electrophoresis of sodium dodecyl sulfate (hereinafter abbreviated as SDS-PAGE). This give a specific activity ^{3. Qx 1 0 6 IU / mg} · protein solution containing 1 4 2 mg of r IFN-§ of the gel filtration process.

 (I) In 36.6 ml (12.0 mg) of the aqueous solution containing rIFN-7 obtained in (上 記) above, 25 mM i-acid buffer solution containing 0.01 M reduced glutathione and 0.0guanidine hydrochloride ^^ ¾6.0) 163.4! 111 was added, and 200 ml of a 0 J 6 mg Zml concentration solution was prepared. This solution was loaded onto a Sephadex G-25 (Pharmacia) column (5 × 60 cm) equilibrated in advance with a 25 mM acetic acid buffer (PH 6.0) containing 0.01 M reduced glutathione, and the same buffer was used. And eluted with, to obtain 220 ml of rIF-7 fraction. The rIF-r concentration of this solution was 0 J47 mg / ml. Then the solution was 4: 4. After aging for 2 days in C, Diaflo P M—

Ultrafiltration using 10 membranes (Namicon ultrafiltration membrane) reduces the volume to 14 ml, and has a protein content of 0.71 Ig / nU and is substantially free of inorganic salts (10 ppm (Ungrooved) A clear rIF-r solution was obtained. The thus obtained high concentration rIF-ri SDS-PAGE converged with the monomer. The specific activity 0 of this rIFN-α was 3.6 × 10 ^s Γ / mg · protein.

 Reference Example 4 Production of high concentration r l F-a aqueous solution substantially free of inorganic salts

 I

Reference Example 2 2 δ mM acetate buffer 5 (pH 6.0) containing 0.01 M reduced glutathione and Q.5 M guanidine hydrochloride in 35 ml (11.48 mg) of the rIFiN ^T -r aqueous solution obtained in (H) ) Was added to add 0.055 mg Zml concentration solution 200 ml

Was pre-equilibrated with 0.01M reduced glutathione solution (PH 6.0). The mixture was loaded on a column G-5 (5 x 60 cm) and developed with a 0.01 M reduced glutathione solution (PH6.0) to obtain an eluted fraction 2335 of rIFN-α. The rlF-a concentration of this solution was 0.046 mg / ml. The solution is then aged at 4 ° C for 1 day, then ultrafiltration of Diaflo-PM-10 membrane, concentrated to 10 ml by the filtration method, and substantially inorganic with a protein concentration of l.OSmgZml. A clear rIFN-aqueous solution without salt (less than 10 ppm) and acetate buffer was obtained. The rIFN- obtained in this manner converged on the monomer by SDS-PAGE. The specific activity of rIF-7 was 3.6 × 10 ⁶ IU / mg · protein. '.

Reference Example 5 Production of Highly Concentrated r I F N -a Aqueous Solution Containing Virtually No Inorganic Salt

 W

Reference Example 1 To 35 ml (11.48 mg) of rIFN-α obtained in (H) was added 16 ml of 25 tnM acetate buffer (pH 6.0) containing 0.01 M reduced glutathione and 0.5 M guanidine hydrochloride. In addition, 200 ml of a Q.057 mg / ml concentration solution was prepared. This solution was previously loaded on a Sephadex G-25 column (5 x 60 cm) equilibrated with a solution containing Q.267M daricin and reduced glutathione (PH 6.0), and developed with the same solution. An eluate fraction of FN—220 ml was obtained. 'The rlF concentration of this solution was Q.046 mg / ml. The solution is then aged for 2 days with 4 parts, concentrated to 9.4 ml by ultrafiltration on a Diaflo PM-10 membrane, and treated with inorganic salts (10%) at a protein content of i.07 mg / ml. ppm) and a clear rIF-r solution without acetic acid buffer. The rl FN- § will converge mono mers in SDS-PAGE, the specific activity was ^{3.62 X 1 0 8 IU / mg} · protein.

 1 2? Reference Example 6 High-concentration death (Cys-Tyr—Cys) containing substantially no inorganic salts

Production of IFX-7 aqueous solution (i) Production of transformants

 I FN-7 expression plasmid PR C 23 / IF I-900 [EPC Publication No.

0 0 8 9 6 7 6 Publication Example 7] was digested with restriction enzymes NdeI and NcoI,

An NdeI-NcoI710 bp DNA fragment (A) containing the 1 F N-a gene portion was collected. On the other hand, plasmid pRC23 was digested with restriction enzymes BglII and EcoRIII, and a DNA fragment (B) of 265 bp containing λPL promoter was isolated. Oligonucleotide adapters that also contain a protein synthesis initiation codon obtained by chemically synthesizing (Α), (Β)

 AATT CAT G CAG GAT C CA

GTAC GT C CTAG GTAT-was bound to the margin of Nde I and EcoR I using Τ4DΝ ligase. Plasmid obtained by treating the obtained DNA fragment with NcoI and Bgigi

 1 2 Q

The expression plasmid pLC2 encoding the polypeptide of Cys-Tyr-Cys deficient IFN-α was constructed by binding to PRC23 / IFI-900 (Fig. 2). Using this plasmid pLC2, Escherichia coli RR I (pRK 248 cIts) was used according to the method of Cohen et al. [Proceedings of National Academy of Sciences, 69, 211 (1972)]. Was transformed to obtain a transformant Escherichia co.li = E. coli RRI (PLC2, pRK248cIts).

(ii) Culture of transformants

Escherichia coli E. coli RRI (pLC2, RK248cIts) containing the plasmid constructed in (i) above was converted to 1% peptotryptone, 0.5% yeast extract, 0.5% saline, 7 gZml Shaking culture was carried out in 50 ml of a liquid medium containing cyclin at 3 δ ° C for 12 hours. Transfer the culture to 2.5fi of M9 medium containing 0.5% casamino acid, 0.5% glucose and 7 g / ml of tetracycline. C The cells were cultured for 4 hours and then at 42 ° C for 3 hours. The cells were collected by centrifugation and stored at 180 ° C.

(Hi) Purification

 Frozen cells obtained in the same manner as in (I) above 7. 7. lg of 22 lg of Tris chloride buffer (PH7.0) containing 7 M guanidine hydrochloride and 2 phenylmethylsulfonyl fluoride After suspending and stirring at 4 ° C for 1 hour, the mixture was subjected to eccentric separation at 10,000 Xg for 30 minutes to obtain 24 ml of the supernatant. The supernatant was diluted with 300 ml of a buffer solution (pH 7.4) consisting of 137 mM sodium chloride, 2.7 mM chloride, 8.1 mM sodium phosphate and 1.5 mM phosphate, and diluted. The mixture was applied to an antibody column (Mor 2-11. Column volume: 15 ml) at a flow rate of 1 mlZ. The column was then washed with 60 ml of 20 mM sodium phosphate buffer containing 0.5 M guanidine hydrochloride (PH7.0), and then 2 OmM sodium phosphate buffer containing 2 M guanidine hydrochloride. This was eluted with 45 ml of (pH 7.0) to obtain a fraction 25πα having antiviral activity. Fifty-five milliliters of this fraction contains 25 mM ammonium acetate buffer (pH 6.0) containing 1 mM ethylenediamine tetraamic acid, 0.15 M sodium chloride, 10 mM cystine and 2 M guanidine hydrochloride in advance. Applied to a column (2.6 × 94 cm) and column volume of 500 ml of Sephacryl S-200 (manufactured by Pharmaceuticals) equilibrated in, and eluted with the same buffer. 0 ml was obtained.

 ]_ twenty three

 Cys— Tyr— Cys missing I F N— obtained from polypeptide Donide ^ 23

(Cys—Tyr—Cys) IFN—a] was 7.0 nig, and the specific activity was 2.7 × 10 ⁶ IU / mg.

(iv) Production of high-concentration aqueous solution Elution fraction containing des (Cys-Tyr-Cys) IFN-γ obtained in (iii) above 2.2 ml (protein concentration 0.18 mg / ml) was diluted with 17.6 ml of 25 mM ammonium acetate buffer (PH6.0) containing 0.5 M guanidine hydrochloride. This solution was loaded onto a Sephadex G-25 column (2.6 × 15 cm) equilibrated with 25 mM ammonium phosphate buffer solution (PH6.0) in advance, and eluted with the same buffer.

 one two Three

23 ml of an eluted fraction of Des (Cys-Tyr-Cs) IF-r from which guanidine hydrochloride was removed (protein concentration O.QISmgZml) was obtained.

 After aging this eluted fraction for 24 hours at 4 ° C,

(25 mm0, Amicon) by ultrafiltration.

(0.2 m) to obtain 0.68 ml of a clear solution. The protein concentration was Q; 41 mg / ml.

 Industrial applicability

 The human IFN-α composition of the present invention, which is substantially free of inorganic salts and frozen or lyophilized under the co-presence of amino acid, can obtain IFN-α during the freezing or freeze-drying operation and storage. (7) Since the activity is extremely low and no turbidity occurs upon dissolution, it can be advantageously used as a pharmaceutical or the like.

Claims

The scope of the claims  1. A human interferon composition frozen or lyophilized under the condition that substantially no inorganic salt is present and an amino acid is present. ,  2. The composition according to claim 1, wherein the amino acid is a monoamino aliphatic amino acid.