MXPA97002578A - Alfa-interferonestable acu solution formulations - Google Patents

Abstract

The present invention discloses stable aqueous solution formulations containing alpha type interferon, e.g., interferon alfa-2a, and interferon alfa-2b, a stabilizer to maintain pH within the range of 4.5 to 7.1, polysorbate 80 as a stabilizer, disodium from adetate as a chelating agent, sodium chloride as a tonicity agent and m-cresol as an antimicrobial preservative and maintaining a high chemical, physical and biological stability of the alpha-type interferon during a Prolonged period of at least 24 months

Description

"FORMULATIONS OF SOLUTION OF ALFA-INTERFERON STABLE AQUEOUS" BACKGROUND OF THE INVENTION This invention relates to formulations of an aqueous stable solution which are free of products derived from human blood serum which maintain high biological activity and high chemical and physical stability of the alpha type interferon, for a prolonged period of time. U.S. Patent Number 4,496,537 discloses formulations of the biologically stable alpha-interferon aqueous solution containing alpha-interferon, human serum albumin, alanine or glycine, water and a stabilizing system to maintain the pH at a value of 6.5 to 8.0. . Human serum albumin ("HSA") acts as a stabilizer for alpha-interferon and prevents the loss of alpha-interferon from the solution by coating and / or absorption of alpha-interferon towards the stainless steel and glass surfaces of containers mixers, process equipment and storage containers. The formulations of the solution containing alpha-interferon and HSA have maintained the chemical and biological stability of alpha-interferon when these solutions have been stored at a temperature of 2 ° C to 8 ° C, for prolonged periods, that is, for more than two years. Recently, global epidemic AIDS has resulted in health registration agencies that require manufacturers to place ads on products, such as alpha-interferon, which contain products derived from human blood, such as HSA. There is a need to reformulate the products of the alpha-type interferon solution to obtain a solution formulation free of products derived from human blood, such as HSA, while maintaining high chemical and physical stability and high biological activity High level of alpha-type interferon in aqueous solution formulations during prolonged storage periods.

SUMMARY OF THE INVENTION The present invention provides a formulation of an aqueous stable solution which maintains high biological activity of the alpha-type interferon and which is free of products derived from human blood comprising: a. from 0.1 x 106 to 100 x 106 IU / milliliter of interferon of the alpha type; b. a stabilizing system to maintain a pH within the range of 4.5 to 7.1; c. an effective amount of a chelating agent; d. an amount of a poly (oxy-1,2-ethanediyl) derivative of mono-9-octadecenoate sufficient to stabilize the alpha-type interferon against loss of the alpha-type interferon; and. an effective amount of a tonicity agent; F. an effective amount of an antimicrobial preservative; and g. an amount of water for injection sufficient to prepare a solution of the ingredients listed above. The present invention provides a formulation of an aqueous stable solution having a high biological activity of the alpha type interferon and which is free of products derived from human blood comprising: a. from 0.1 x 106 to 100 x 106 IU per milliliter of alpha-type interferon. b. a stabilizing system sufficient to maintain the pH of the solution within the range of 4.5 to 7.1; c. from about 0.01 to 1 milligram per milliliter of disodium dihydrogen ethylenediaminetetraacetate. d. from about 0.01 to 1 milligram per milliliter of a poly (oxy-1,2-ethanediyl) derivative of sorbitan mono-9-octadecenoate; and. from about 1 to 9 milligrams per milliliter of sodium chloride; F. an effective amount of an antimicrobial preservative selected from m-cresol, phenol, methylparaben, propylparaben or mixtures thereof; and g. water for injection in sufficient quantity to reach one milliliter. In a preferred aspect, the present invention provides a formulation of an aqueous stable solution having a high biological activity of the alpha-type interferon, and which is free of products derived from human blood comprising: mg / ml a. Alpha-2 interphone 5 x 106 to 50 x 106 IU b. Sodium Phosphate Dibasic Anhydrous 1.8 c. Monobasic Monobasic Sodium Phosphate 1. 3 d. Disodium Dihydrogen Ethylenediaminetetraacetate 0.1 e. Polysorbate 80 0.1 f. Methylparaben 1.2 g. Propilparaben 0.12 h. Sodium chloride; 7.5 and i. Water for Injection sufficient quantity to reach 1 milliliter In another preferred aspect, the present invention further provides a formulation of an aqueous stable solution having a high biological activity of the alpha-type interferon and which is free of products derived from human blood comprising: mg / ml a. Interferon Alfa-2 5 x 106 to 50 x 106 IU b. Sodium Phosphate Dibasic Anhydrous 1.8 c. Monobasic Monobasic Sodium Phosphate 1.3 d. Disodium Dihydrogen Ethylenediaminetetraacetate 0.1 e. Polysorbate 80 0.1 f. m-Cresol 1.5 g. Sodium chloride; 7.5 and h. Water for Injection sufficient quantity to reach 1 milliliter The present invention also provides a process for preparing a formulation of the stable aqueous solution having high biological activity of the alpha-type interferon and which is free of products derived from human blood comprising mixing an effective amount of the alpha-type interferon with a stabilizing system capable of maintaining a pH within the range of 4.5 to 7.1, a chelating agent, a poly (oxy-1,2-ethanediyl) derivative of sorbitan mono-9-octadecenoate, a tonicity agent, a preservative antimicrobial in sufficient quantity to form a solution. In a preferred aspect of the process of the present invention, the solution is prepared and maintained essentially free of dissolved oxygen and a top space of inert atmosphere above the solution is maintained at a value of less than about 4 volume percent by volume. oxygen.

DETAILED DESCRIPTION We have selected specific amounts of a set of specific ingredients that have allowed us to develop a formulation of an aqueous solution of alpha-type interferon that does not contain human serum albumin, but nevertheless maintains high biological and physical chemical stability for interferon-type alpha during storage at a temperature of 2 ° C to 8 ° C for prolonged periods of at least 24 months. The term "free of products derived from human blood" as used herein with reference to the formulations of the present invention means that products derived from human blood, such as HSA, are not used in the preparation of the formulations of the solution. of the present invention. The term "high chemical stability" as used herein with reference to the alpha type interferon used in the formulations of the present invention, means that the alpha type interferon maintains at least 85 percent, preferably 85 percent to 100 percent of its chemical integrity during storage at a temperature of 2 ° C to 8 ° C for at least 24 months. See Tables 1 and 2. Chemical integrity is determined by measuring the protein content in an HPLC assay such as that disclosed by T.L. Nagabhushan, et al., In an article called "Characterization of Genetically Engineered ALFA-2 Interferon", pages 79 to 88 that appears in Interferon Research Clinical Application and Regulatory Consideration, Zoon et al., Editors, Elsevier Science Publishing Co. Inc. 1984 ( See the results in Tables 1 to 4). The term "high biological stability" as used herein with reference to the alpha-type interferon used in the formulations of the present invention means that the alpha-type interferon in the formulation maintains at least 75 percent, preferably at least less 85 percent and more preferably 90 percent to 100 percent of their biological activity during storage at 2 ° C to 8 ° C for at least 24 months (see results in Tables 1 to 4) as measured in the method of normal inhibition of the cytopathic effect (CPE) of a virus such as the method disclosed by WP Protzman et al., In J. Clinical Microbiology, (1985), 22, 596-599.

The term "high physical stability" as used herein with reference to the alpha type interferon used in the formulations of the present invention, means that the formulation of the present invention remains crystalline, i.e., does not exhibit turbidity or visible particulate matter (i.e., particles greater than about 60 to 70 microns in diameter) during storage at 2 ° C to 8 ° C during at least 24 months. See Tables 1, 2 and 3. The results listed in Tables 1, 2 and 3 are surprising since most formulations of the solution containing protein products, such as alpha-type interferon tend to develop material in visually observable particles (ie particles having diameters greater than 60 to 70 microns) during prolonged storage at a temperature of 2 ° C to 8 ° C. The test method used to determine the particulate matter in the formulation of the solution of this invention (see Tables 1 to 4) is described in The United States Pharmacopeia / The National Formulary USP 23 / NF 18, published by United States Pharmacopeia Convention, Inc. (1995), of Rockville, Maryland; see the Physical Test < 788 > at pages 1813 to 1816. The method used to determine the visual description of the formulations of the solution of this invention is also described in USP 23 as "General Requirement Test and Assays < 1 > Injections" on pages 1650 a 1652. We have found that by adding a chelating agent to the formulations of the present invention, we have been able to avoid visible particulate matter. Typical suitable chelating agents include disodium dihydrogen ethylenediamine tetraacetate (EDTA or edetate disodium) or citric acid. The use of edetate disodium is preferred. Although we do not wish to be bound by any theory, it is believed that the disodium of edetate is effectively complexed with trace amounts of the metal cations, such as Zn ^ "1", Fe ^ +, Cu ^ + or A1 -3+, whose ions may be present in excipients and packaging components, e.g., rubber plugs or gaskets. Since the disodium of edetate has greater affinity for these metal cations than the alpha-type interferons, the interaction between the metal cations and the alpha-type interferon which results in the formation of insoluble complexes (in the form, for example , of a visible particulate matter) and loss of activity, are avoided of course. The effective amount of the chelating agent falls within the range of 0.01 to 1 milligram per milliliter based on 0.1 x 106 to 100 x 106 International Units ("UI") of alpha type interferon per milliliter. Preferably - Il ¬ use 0.1 milligram of disodium of edetate for 5 x 106 to 50 x 106 IU of interferon alfa-2. Suitable stabilization systems for the formulations of the present invention are those that maintain the pH of the aqueous solution formulation within the range of 4.5 to 7.1, preferably 6.5 to 7.1 and especially preferred to 6.8. The use of a stabilizing system of sodium phosphate dibasic and sodium phosphate monobasic is preferred. Normally, from 0.005 to 0.1 molar of the stabilizing agent of the sodium phosphate monobasic system / preferred dibasic stabilizer is used for a formulation containing 0.1 x 106 to 100 x 10ß IU of the alpha type interferon per milliliter. Other stabilizer systems suitable for maintaining the desired pH range of 4.5 to 7.1 include sodium citrate / citric acid and sodium acetate / acetic acid. The tonicity agent useful in the present invention is any agent capable of rendering the formulations of the present invention iso-osmotic with human serum. Typical suitable tonicity agents include sodium chloride, mannitol, glycine, glucose and sorbitol. The use of sodium chloride as a tonicity agent is preferred.

The amount of the tonicity agent used is within the range of 1 to 10 milligrams per milliliter when the formulation of the present invention contains 0.1 x 10 6 to 100 x 10 7 IU of the alpha type interferon per milliliter. The use of 7.5 milligrams per milliliter of sodium chloride will be preferred for 5 x 10 ^ to 50 x 10 ^ IU of the alpha type interferon per milliliter, in the formulations of the present invention. The poly (oxy-1, 2-ethanediyl) derivatives of sorbitan mono-9-octadecenoate, such as polysorbate 80 or polysorbate 20 are useful as a stabilizing agent to prevent absorption of alpha-type interferon proteins, such as interferon alfa-2b on stainless steel and glass surfaces of the equipment used to make the indicated formulations containing the alpha-type interferon. The amount of polysorbate 20 or 80 effective in the formulation of this invention falls within the range of 0.01 to 1.0 milligram per milliliter for a formulation containing 0.1 x 10 ^ to 100 x 106 IU of alpha-type interferon per milliliter. The use of polysorbate 80 is preferred. The use of 0.1 milligram per milliliter of polysorbate 80 is especially preferred in all formulations of the solution of the present invention. When alpha-type interferon concentrations, such as interferon alfa-2, are less than approximately 15 x 10 ^ IU / milliliter, v, gr, 6 x 106 IU / milliliter, the loss of activity due to the absorption of alpha -interferon in the absence of polysorbate 80 significantly decreases the biological activity of the formulation. Surprisingly we have found that polysorbate 80 prevents the loss of interferon alfa-2b and allows the systematic delivery of interferon alfa-2b without loss of biological activity. In the course of developing the formulation of the present invention, we have surprisingly found that polysorbate 80 provided superior chemical and biological stability to interferon alpha-2b compared to the other nonionic surfactants, e.g., Pluronic F127 and Pluronic F-68. The amount of the alpha-type interferon useful in the formulation of the present invention falls within the range of 0.1 x 106 to 100 x 106 IU per milliliter, preferably from 5 x 10 ^ to 50 x 10 ^ iu per milliliter. The term "alpha-type interferon" as used herein means the family of highly homologous species-specific proteins that inhibit viral duplication and cell proliferation and modulate the immune response. Typical alpha-type interferons include interferon alpha-2a such as ROFERON A, an interferon alfa-2a obtainable from Hoffman-La Roche, from Nutley, NJ, interferon alfa-2b, such as INTRON A interferon alfa-2b obtainable from Schering Corporation, from Kenilworth, NJ, interferon alfa-2c, such as BEROFOR interferon alfa-2c obtainable from Boehringer Ingelheim Pharmaceutical, Inc., of Ridgefield, CT, interferon alfa-nl, a purified mixture of natural alpha-interferons, such as SUMIFERON obtainable from Sumitomo, Japan or as WELLFERON the interferon alfa-nl obtainable from The Wellcome Foundation Ltd. of London, Great Britain, or in consensus of alpha-interferon obtainable from Amgen, Inc. of Newbury Park, California or the interferon alfa-n3, a mixture of natural alpha-interferons that are produced by Interferon Sciences and obtainable from Purdue Frederick Co. of Norwalk, CT, under the name ALFERON. The use of interferon alfa-2a or alpha-2b is preferred. The use of interferon alfa-2b is especially preferred. Antimicrobial preservatives that have been found to be useful in the present invention include m-cresol, phenol and methyl paraben and propylparaben and mixtures of the above-numbered preservatives, e.g., mixtures of phenol-methylparaben. The effective amount of m-cresol found to be useful in the present invention falls within the range of 0.5 to 2 milligrams per milliliter for a formulation containing 0.1 x 106 to 100 x 106 IU / milliliter of alpha-type interferon. . It is preferred to use 1.5 milligrams per milliliter of m-cresol for a formulation containing 5 x 10 ^ to 50 x 10 ^ IU per milliliter of interferon alfa-2b. The effective amount of phenol found useful falls within the range of 0.5 to 5 milligrams per milliliter for a formulation of the solution containing 0.1 x 106 to 100 x 106 IU per milliliter of alpha-type interferon. The effective amount of methylparaben is within the range of 0.6 to 1.8 milligrams per milliliter, and the amount of propylparaben is within the range of 0.06 to 0.18 milligram per milliliter when the formulation of the present invention contains 0.1 x 10 ^ to 100 x 10 ^ IU / milliliter of alpha type interferon. It is preferred to use 1.2 milligrams per milliliter of methylparaben in combination with 0.12 milligram per milliliter of poliparaben when the formulation of the present invention contains from 0.1 x 10 ^ to 100 x 10 ^ IU per milliliter of interferon alfa-2b. The use of m-cresol as an antimicrobial preservative is especially preferred. The water used for the preparation of the formulations of the present invention is preferably water for injection.

During the course of the development of the aqueous solution formulations of the present invention which would maintain a high biological activity as well as high chemical and high physical stability of the alpha type interferon through a prolonged storage period without employing HSA as a stabilizer we have identified that the amount of a poly (oxy-1, 2-ethanediyl) derivative of sorbitan mono-9-octadecenoate, such as polysorbate 80 required to act as a stabilizing agent for alpha-type interferon, had a direct effect in the effective amount of the antimicrobial preservative that could be added to the aqueous solution formulation in order to provide the appropriate antimicrobial protection for the formulation, in adance with the different health registration requirements without causing undesirable turbidity formation in the solution . Thus, when present in the formulations of the present invention, the preferred stabilizing agent, polysorbate 80, in the preferred effective amount of 0.1 milligram per milliliter, the effective amount of the preferred antimicrobial preservative, e.g. , m-cresol that could be added without causing turbidity of the formulation was found to be critical. For example, if the amount of m-cresol added to a formulation containing 0.1 milligram per milliliter of polysorbate 80, as shown in Example 3 is increased to more than 1.75 milligrams per milliliter, turbidity is observed. A similar turbidity problem was observed when the amount of polysorbate 80 in the resulting formulation was varied from 0.01 to 1 milligram per milliliter. No turbidity was observed when an amount of 1.75 milligrams per milliliter or less, preferably about 1.5 milligrams per milligram of m-cresol, was added to a formulation prepared in adance with the procedures of Example 3 containing 0.1 milligram per milliliter of polysorbate 80 This criticality was also observed with parabens and phenol when antimicrobial preservatives were used. For formulations of the present invention containing from 0.01 to 1 milligram per milliliter of polysorbate 80, the effective amount of methylparaben should be no greater than about 1.2 milligrams per milliliter when used with 0.12 milligram per milliliter of propylparaben to prevent turbidity, and the effective amount of phenol (when used instead of parabens) should be within the range of 0.5 to less than about 4 milligrams per milliliter, to avoid clouding.

Alpha-type interferon formulations are useful for the treatment of a variety of disease states, such as renal cell carcinomas, AIDS-related Kaposi's sarcoma, chronic and acute hepatitis B, non-chronic hepatitis B, and non-B / C hepatitis. acute The formulations of the present invention are useful for treating these disease states, preferably as injectable aqueous solutions.

EXAMPLES The following non-limiting examples illustrate the preparation of the aqueous solutions of alpha-type interferons. The procedures listed after the Example are used to prepare the formulations of the present invention of Examples 1 to 5.

EXAMPLE 1 Active Substance: Interferon alfa-2b from 0.1 x 106 to 100 x 106 IU / ml * Stabilizing Agent: Sodium Phosphate (monobasic / dibasic) from 0.005 to 0.1 M Chelation Agent: Edetate Disodium from 0.01 to 1 mg / ml Stabilizer: Polysorbate 80 from 0.01 to 1 mg / ml Tonicity Adjustment Agent: Sodium Chloride from 1 to 9 mg / ml Antimicrobial Preservative: m-Cresol from 0.5 to 1.75 mg / ml or Phenol from 0.5 to < 4 mg / ml or Methylparaben from 0.6 to 1.2 mg / ml Propilparaben from 0.06 to 0.12 mg / ml Solvent: Water for Injection in sufficient quantity to reach 1 ml * IU - International Units EXAMPLE 2 Interferon alfa-2b 10 xlO6 IU / ml Sodium Phosphate Dibasic Anhydrous 1.8 mg / ml Sodium Phosphate Monobasic Monohydrate 1.3 mg / ml Edetate Disodium 0.1 mg / ml Polysorbate 80 0.1 mg / ml Methylparaben 1.2 mg / ml Propilparaben 0.12 mg / ml Sodium Chloride 7.5 mg / ml Water for Injection sufficient amount to reach 1 ml EXAMPLE 3 Interferon alfa-2b 10 x 106 IU / ml Sodium Phosphate Dibasic Anhydrous 1.8 mg / ml Sodium Phosphate Monobasic Monohydrate 1.3 mg / ml Edetate Disodium 0.1 mg / ml Polysorbate 80 0.1 mg / ml m-Cresol 1.5 mg / ml Sodium Chloride 7.5 mg / ml Water for Injection sufficient amount to reach 1 ml The stability data in Examples 2 and 3 are summarized in Tables 1 and 2, respectively.

EXAMPLE 4 The formulation of Example 3 was prepared with 6 x 10 6 IU / ml of interferon alfa-2b in accordance with the manufacturing method detailed below using nitrogen spray from the solution and not maintaining more than about 4 volume percent of the oxygen in the upper space. Small bottles containing a volume of 3 milliliters of solution were stored at temperatures of 30 ° C, 25 ° C and 4 ° C. The results are summarized in Table 3. EXAMPLE 5 The formulation of Example 4 was prepared in accordance with the manufacturing method detailed below in all details, except that no nitrogen was sprayed through the solution or placed above the same and the volume of oxygen in the upper space was ~ 20 percent by volume as found in the ambient air. Small bottles containing a volume of 3 milliliters of solution were stored at temperatures of ° C, 25 ° C and 4 ° C, the results are summarized in Table 4. Similar results are expected if the interferon alfa-2b in Examples 1 to 5 is substi- tuted from an equivalent amount of Roferon A, Wellferon or Sumiferon , in interferon alfa.

TABLE 1 Stability Data of Interferon Alfa-2b in Example 2 Tempe Time- Antiviral Assay Protein Content (CPE) (HPLC Assay) (month) (° C) (xl06IU / ml) (% LS) ( mcg / ml) (% Initial) Initial 10.0 100 42.5 100 3 4 9.0 90 42.4 100 6 4 10.0 100 41.8 98 9 4 10.0 100 43.2 102 12 4 10.0 100 44.3 104 18 4 9.8 98 41.5 98 24 4 10.0 100 39.5 93 Matter in Particles Description (particles / container) _ > 10μ > 25μ > 50μ 40 3 1 CCS1 16 13 11 CCS 8 3 1 CCS 52 3 0 CCS 17 4 2 CCS 6 1 0 CCS 5 1 0 CCS * ccs - colorless crystalline solution, essentially free of visible particles.

TABLE 2 Stability Data of Interferon Alfa-2b in Example 3 Tempe Time- Antiviral Assay Protein content (CPE) (HPLC Assay) (month) (° C) (xl06IU / ml) (% LS) ( mcg / ml) (Initial) Initial 10.3 103 37.9 100 1 4 10 100 38.2 101 3 4 10 100 38.9 103 6 4 10 100 40.0 105 9 4 10 100 37.1 97.9 12 4 10 100 36.6 96.6 4 10 100 36.1 95.3 Matter in Particles Description (particles / packaging) > 10μ > 25μ > 50μ 68 4 3 CCS * 142 24 23 CCS 311 63 35 CCS 206 17 16 CCS 211 109 50 CCS 300 65 12 CCS 123 8 6 CCS * ccs - colorless crystal solution, essentially free of visible particles TABLE 3 Data of Stability of Interferon Alfa-2b in Example 4 Tempe Time- Position Test Antiviral jar ration (CPE) (month) (° C) small * (xl06IU / ml) (% LS) Initial UP 6.00 100 INV 6.00 100 UP 6.00 100 INV 6.00 100 25 UP 6.00 100 INV 6.00 100 UP 6.00 100 INV 6.00 100 25 UP 5.58 92.3 INV 6.00 100 12 UP 6.00 100 INV 6.00 100 (Table 3 below) Protein Content m-Cresol Assay (HPLC Assay) (mcg / ml) (Initial%) (mg / ml)% LS pH . 7 100 1.47 98.0 6.91 24. 8 96.5 1.47 98.0 6.90 24. 8 96.5 1.47 98.0 6.90 23. 5 91.4 1.46 97.3 6.88 24. 2 94.2 1.46 98.7 6.87 21. 7 84.4 1.43 95.3 6.88 21. 7 84.4 1.47 98.0 6.88 24. 6 95.7 1.46 97.3 6.84 24. 3 94.6 1.45 96.7 6.84 . 5 79.8 1.45 96.7 6.85 . 4 79.4 1.44 96.0 6.85 23. 4 91.0 1.47 98.0 6.84 23. 4 91.0 1.46 97.3 6.84 * UP - Vertical INV - Inverted TABLE 3 (continued) Time Tempe- Position Matter in Particles Fracture of the bottle (No. of Particles / Small Cryption1 small vial) (month] (° C)> 10μm _> 25μm> 50μm Initial 23 CCS ' 1 30 UP 109 61 16 CCS INV 55 11 2 CCS UP 29 2 0 CCS INV 59 23 2 CCS 25 UP 141 76 17 CCS INV 49 16 3 CCS UP 59 21 3 CCS INV 68 20 4 CCS 25 UP 38 4 0 CCS INV 57 4 0 CCS 12 UP 21 2 0 CCS INV 18 2 0 CCS * CCS - Colorless crystalline solution, essentially free of visible particles.

TABLE 4 Stability Data of Interferon Alfa-2b in Example 5 Time Tempe- Position Test Activiral jar ration (CPE) (month) (° C) small * (xl06IU / ml) (% of L.S.) Initial 6.00 100 1 30 U UPP 6 6..0000 100 I INNVV 6 6..0000 100 3 4 U UPP 6 6..0000 100 INV 6.00 100 25 U UPP 6 6..0000 100 INV 6.00 100 6 4 U UPP 6 6. .0000 100 INV 6.00 100 25 U UPP 6 6..0000 100 INV 6.00 100 12 4 U UPP 7 7..5566 126 I INNVV 7 7..0000 117 (Table 4 (continued) Protein Content m-Cresil Assay HPLC Assay) (mcg / mL) (Initial%) (mg / mL)% LS pH . 5 100 1.47 98.0 6.85 19. 5 76.5 1.49 99.3 6.82 19. 5 76.5 1.50 100 6.83 24. 0 94.1 1.43 95.3 6.81 24. 0 94.1 1.43 95.3 6.82 . 2 79.2 1.39 92.7 6.82 19. 6 76.9 1.41 94.0 6.82 24. 6 96.5 1.47 98.0 6.82 24. 6 96.5 1.47 98.0 6.83 17. 2 67.5 1.47 98.0 6.83 16. 1 63.1 1.48 98.7 6.84 23. 3 91.4 1.58 105 6.88 23. 2 91.0 1.41 94.0 6.88 (Table 4 (continued) Time Tempe- Position Matter in Particles Fracture of the bottle (No. of particles / cryp- small small vial) tion (month) (° C) > 10μm > 25μm > 50μm Initial 144 CCS1 1 30 UP 89 3 1 CCS INV 64 1 0 CCS 3 4 UP 39 1 0 CCS INV 77 19 6 CCS 3 25 UP 57 1 0 CCS INV 140 24 1 CCS 6 4 UP 66 1 0 CCS INV 220 87 27 CCS 25 UP 92 3 0 CCS INV 241 5 0 CCS 12 4 UP 63 1 0 CCS INV 119 6 1 ees * CCS - Colorless crystalline solution, essentially free of visible particles.

Manufacturing Method for Examples 1 to 5 A. Mixing of Formulations of the Aqueous Solution Containing Paraben as Samples in Example 2. 1. About 80 percent water for injection is charged at a temperature greater than 70 ° C in a jacketed mixing vessel equipped with a stirrer. 2. Approximately 30 percent of the water for injection is separately charged into another appropriate container. It cools and maintains the water temperature between 20 ° C and 25 ° C. Begin spraying and placing above the water that will be used to bring the batch to the final volume with filtered nitrogen to maintain a dissolved oxygen level of or less than 0.25 part per million. 3. Methylparaben and propylparaben are charged and dissolved in the mixing vessel in step 1, while the temperature of the solution is maintained between 70 ° C and 80 ° C. 4. The solution is cooled in step 3 to a temperature between 20 ° C and 25 ° C. It is sprayed and placed over the solution with filtered nitrogen. A level of dissolved oxygen at or below 0.25 part per million is maintained. 5. Charge and dissolve the following ingredients in the solution in step 4, while maintaining the spraying and nitrogen placement: Dibasic sodium phosphate anhydrous Sodium phosphate monobasic monohydrate Edetate disodium Sodium chloride 6. Sodium phosphate discontinue the nitrogen spray from the solution in step 5. Keep the nitrogen above the mixing container. 7. Charge and dissolve the polysorbate 80 in approximately 50 milliliters of water for injection for a one liter size batch in a separate vessel, transfer the polysorbate 80 solution to the solution in step 6. 8. Check the pH of the solution. It should be between 6.6 and 7.0. No pH adjustment is required. 9. The bulk drug solution of interferon alfa-2b is loaded into the solution in step 8, while mixing. 10. Water for injection that has been sprayed with nitrogen (from step 2) is added to bring the batch to the final volume. The solution is stirred gently until homogeneous. 11. It is filtered aseptically to the solution through a sterilized filter that has been washed and tested for its integrity. The sterilized solution is collected in a sterilized filling container that has been filled with sterile filtered nitrogen. The filter is tested for integrity after filtration. 12. The filling container is filled in step 11 with sterile filtered nitrogen and sealed.

B. Mixing the Formulations of the Aqueous Solution Containing m-Cresol, as Shown in Example 3.

The manufacturing process used to prepare the aqueous solution containing m-cresol as a preservative (as shown in Example 3) is exactly the same as described above with the exception that the temperature of the Solution in Step 3 is maintained between 20 ° C and 25 ° C and the m-cresol is loaded after Step 6.

C. Mixing the Formulations of the Aqueous Solution of Alpha-Interferon Free of HSA Under Air Environment The manufacturing process used to prepare the aqueous HSA-free alpha-interferon formulations of Examples 1 to 4 was used to prepare the formulations, such as that of Example 5 with the exception that all steps were carried out under air ambient. No nitrogen was sprayed through the solution or placed above and the ambient air (which normally contains about 20 volume percent oxygen) occupied the volume of the upper space. In order to maintain the elevated chemical, physical and biological stability, it is preferred that the water used to prepare the aqueous solution of alpha-interferon as well as the aqueous solution of alpha-interferon formed in this way be essentially free of dissolved oxygen and the aqueous solution prepare and store with a top space of an inert atmosphere, such as nitrogen that does not contain more than 4 volume percent oxygen. By the term "essentially free of dissolved oxygen" as used herein, is meant an oxygen level of no more than about 0.25 part per million at a water temperature of about 20 ° C to 25 ° C. Normally, this preferred dissolved oxygen level of 0.25 part per million is conveniently achieved by spraying an inert atmosphere, eg, nitrogen gas into the water used to prepare the aqueous solutions (maintained at a temperature of about 20 ° C to 25 ° C). ° C) for a sufficient period of time (eg, at about 30 minutes) to decrease the dissolved oxygen to a value of no more than about 0.25 part per million. Spraying is continued through the manufacturing process to keep the dissolved oxygen level at 0.25 part per million. We have found that the aqueous formulations of the present invention have a dissolved oxygen level of one part per million and an oxygen content in the upper space of 7 volume percent, demonstrated significantly greater losses of chemical stability of alpha-interferon after of three months of storage at 25 ° C, compared to a similar aqueous formulation having the preferred dissolved oxygen level of 0.25 parts per million and an oxygen content in the upper space of 4 volume percent stored under the same conditions . A side-by-side comparison of the stability data of the interferon alpha-2b solution shown in Tables 3 and 4 shows that there is no significant stability difference between the aqueous solution formulations of the present invention that were prepared under of nitrogen / low oxygen used in Example 4 and those prepared according to Example 5 under ambient air during 12 months storage at 4 ° C. In contrast, a comparison of the stability of interferon alfa-2b in solutions of Examples 4 and 5 stored at higher temperatures e.g., from 25 ° C to 30 ° C shows the protective effect achieved by the preferred use (more of nitrogen spraying to effect low dissolved oxygen levels in the aqueous solution while simultaneously maintaining an oxygen content in the space above a value no greater than about 4 volume percent The formulation of the aqueous solution of the The present invention can be stored in any of the appropriate washed and sterilized filling containers or in a container such as small glass jars of 2 milliliter capacity or 5 milliliters of Type I with gray butyl rubber closures. Aqueous of the present invention can also be stored in multi-dose pre-filled syringes, such as those useful for delivering drug solutions such as insulin. Typical suitable syringes include systems comprising a pre-filled small vial attached to a pen-type syringe such as Novolet Novo Pen obtainable from Novo Nordisk. Typical suitable systems include a pre-filled pen type syringe that allows for easy self-injection by the user as well as exact reproducible dose rates. The formulations of the aqueous solutions of the present invention, as presented in the Examples, can also be lyophilized to form a powder for reconstitution. Interferon powder of the lyophilized alpha type is expected to maintain its chemical and biological stability when stored at a temperature of 2 ° C to 8 ° C for at least 2 years.

Claims (13)

R E I V I N D I C A C I O N E S:

1. An aqueous stable formulation having high biological activity of the alpha type interferon and which is free of products derived from human blood comprising: a. from 0.1 x 106 to 100 x 106 IU / milliliter of alpha-type interferon; b. a stabilizing system to maintain a pH within the range of 4.5 to 7.1. c. an effective amount of a chelating agent; d. an amount of a poly (oxy-1, 2-ethanediyl) derivative of sorbitan mono-9-octadecenoate, sufficient to stabilize the alpha-type interferon against loss of the alpha-type interferon; and. an effective amount of a tonicity agent; F. an effective amount of an antimicrobial preservative; and g. an amount of water for injection sufficient to prepare a solution of the ingredients listed above.

2. The composition according to claim 1, wherein the stabilizing system is sodium dibasic phosphate and monobasic sodium phosphate.

3. The composition according to claim 1, wherein the chelating agent is disodium of edetate or citric acid.

4. The composition according to claim 1, wherein the tonicity agent is sodium chloride. The composition according to claim 1, wherein the preservative is selected from m-cresol, phenol, methylparaben, propylparaben or mixtures thereof. 6. The composition according to claim 1, wherein the alpha type interferon is interferon alpha-2. 7. A formulation of a stable aqueous solution having high biological activity of interferon alfa-2 and which is free of human serum albumin comprising: mg / ml a. Interferon alfa-2 5 x 106 to 50 x 106 IU b. Sodium Phosphate Dibasic Anhydrous 1.8 c. Monobasic Monobasic Sodium Phosphate 1.3 d. Disodium Dihydrogen Ethylenediaminetetraacetate 0.1 e. A Poly (Oxi-1, 2- Ethandiyl) Derivative of Sorbitan Mono-9-Octadecenoate 0.1 f. Methylparaben 1.2 g. Propilparaben 0.12 h. Sodium chloride; and 7.5 i. Water for Injection sufficient quantity to reach 1 ml 8. A formulation of a stable aqueous solution having high biological activity of interferon alfa-2 and which is free of human serum albumin comprising: mg / ml a. Interferon alfa-2 5 x 106 to 50 x 106 IU b. Sodium Phosphate Dibasic Anhydrous 1.8 c. Monobasic Monobasic Sodium Phosphate 1.3 d. Disodium Dihydrogen Ethylenediaminetetraacetate 0.1 e. A Poly (Oxi-1, 2- Ethandiyl) Derivative of Sorbitan Mono-9-Octadecenoate 0.1 f. m-Cresol 1.5 g. Sodium chloride; and 7.5 h. Water for Injection sufficient quantity to reach 1 milliliter 9. An article of manufacture comprising a packing material and the formulation of any of the preceding claims, wherein the packaging material is a small multi-dose glass bottle. 10. A manufacturing article comprising a pre-filled syringe containing an effective amount of the formulation of any of the preceding claims. 11. A manufacturing article comprising a packing material and the formulation of any of the preceding claims, wherein the packaging material is a small single-dose bottle. 12. A process for preparing a stable aqueous formulation having high biological activity of the alpha-type interer and which is free of products derived from human blood comprising mixing an effective amount of the alpha-type interferon with a stabilizing system capable of maintaining the pH within the range of 4.5 to 7.1, and a chelating agent, a poly (oxy-1, 2-ethanediyl) derivative of sorbitan mono-9-octadecenoate, a tonicity agent, an antimicrobial preservative and sufficient water to produce an aqueous solution. 13. The process according to claim 12, wherein the aqueous solution is prepared and maintained essentially free of dissolved oxygen.