we have selected specific amounts of a specific set of ingredients that have allowed us to develop an aqueous pegylated alpha - type interferon solution formulation which does not contain benzyl alcohol yet maintains high chemical , biological and physical stability for the pegylated alpha - type interferon for extended periods of time . the term “ free of benzyl alcohol ” or “ benzyl alcohol free ” as used herein in reference to the formulations of the present invention means that no benzyl alcohol is used in the preparation of the solution formulations of the present invention . the buffer systems suitable for the formulations of the present invention are those which maintain the ph of the aqueous solution formulation in the range of 5 . 5 to 6 . 5 , preferably 5 . 8 - 6 . 2 and most preferably 6 . 0 . the use of a buffer system of sodium acetate / acetic acid is preferred . other suitable buffer systems to maintain the desired ph range of 5 . 5 to 6 . 5 include sodium citrate / citric acid and sodium phosphate dibasic and sodium phosphate monobasic . 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 . use of sodium chloride as a tonicity agent is preferred . the sorbitan mono - 9 - octadecenoate poly ( oxy - 1 , 2 - ethanediyl ) derivative polysorbate 20 is useful as a surfactant to prevent adsorption of the pegylated alpha - type interferon proteins such as 40 kda branched pegylated alpha - 2a interferon ( pegasys ®) onto the stainless steel and glass surfaces of the equipment used to make the indictable formulations containing pegylated alpha - type interferon . the amount of polysorbate 20 is in the range of 0 . 005 to 0 . 5 percent by weight , preferably 0 . 02 percent by weight for a formulation containing 0 . 1 0 . 5 mg / ml pegylated alpha - type interferon . surprisingly , we have found that polysorbate 20 prevents loss of pegylated alpha - 2a interferon and allows systemic delivery of the pegylated alpha - 2a interferon without loss of biological activity . in the course of development of the formulation of the present invention , we surprisingly found that polysorbate 20 provided superior chemical and biological stability to a pegylated alpha - 2a interferon in the presence of l - methionine ( replacing benzyl alcohol ) compared to other sorbitan mono - 9 - octadecenoate poly ( oxy - 1 , 2 - ethanediyl ) derivative surfactants , e . g ., polysorbate 80 . similar chemical and biological stability is achieved when poloxamer 188 is used instead of polysorbate 20 at the same concentration . the amount of pegylated alpha - type interferon useful in the formulation of the present invention is in the range of 0 . 1 to 0 . 5 mg / ml . as used herein the term “ pegylated alpha - type interferon ” means covalent conjugates of one or more polyethylene glycol ( peg ) molecules and one or more alpha - type interferon molecules . preferred conjugates for use in the formulations of the invention have one to four peg molecules per interferon molecule , and more preferably , the conjugates are between a single peg molecule and a single interferon molecule . the pegylated interferon may comprise a single positional isomer or a mixture of conjugate positional isomers , e . g , the peg molecules are covalently attached to different amino acid residues on the individual interferon molecules . for example , u . s . pat . no . 5 , 951 , 974 describes the preparation of mixtures of peg - interferon alpha conjugate positional isomers in which some of the isomers are conjugates between peg and a histidine residue of the interferon molecule , other isomers in the mixture are conjugates between peg and an interferon lysine residue and still other isomers are conjugates between peg and the amino terminus of the interferon molecule . the peg molecules in the conjugates may have different molecular weights . preferably , the peg molecule has an average molecular weight of 40 , 000 . in a particularly preferred embodiment , the conjugates are prepared using a branched peg 40000 , i . e ., which means the peg molecules in the conjugates will have an average molecular weight of about 40 , 000 . the interferon portion of the pegylated alpha - type interferon conjugates used in the present invention may be any naturally - occurring or recombinant interferon alpha known to those skilled in the art . natural and recombinant alpha - interferons that may be used in the formulations of the invention include interferon alpha - n1 ( e . g ., surniferon ®, surnitomo ®), interferon alpha - n3 , interferon alpha - 2a ( roferon ® a , hoffmann - laroche , inc .) interferon α - 2b ( intron ® a , schering - plough corp . ), interferon alpha - 2c ( berofor ®, boehringer ingelheim , inc . ), and consensus interferon ( infergen ®, intermune , inc .). preferred interferons are interferon alpha - 2a and interferon alpha - 2b . most preferably , interferon alpha - 2a is used to prepare the active ingredient of the formulations of the present invention . conjugation of the peg and interferon molecules may be performed by any conjugation reaction known to those skilled in the art , e . g ., as described in u . s . pat . nos . 5 , 612 , 460 , 5 , 711 , 944 and 5 , 951 , 974 . preferably , the peg molecule is covalently attached to the interferon molecule with a urethane bond . the most preferred pegylated alpha - type interferon for use in the formulations of the invention is a branched peg 40000 - interferon alpha - 2a . the water used for preparation of the formulations of the present invention is preferably water for injection . during the course of development of the aqueous solution formulations of the present invention that would maintain high biological activity as well as high chemical and high physical stability of the pegylated alpha - type interferon over an extended storage period without employing benzyl alcohol as a stabilizer , we identified that l - methionine can only successfully replace benzyl alcohol as stabilizer when either polysorbate 20 or poloxamer 188 is used as surfactant . pegylated alpha - type interferon formulations are useful for treatment of a variety of disease states such as renal cell carcinomas , aids - related kaposi &# 39 ; s sarcoma , chronic and acute hepatitis b , chronic and acute non - a , non - b / c hepatitis . the formulations of the present invention are useful in treating these disease states preferably as injectable aqueous solutions . the following non - limiting examples illustrate the preparation of the aqueous solutions of pegylated alpha - type interferons . commercial pegasys drug substance ( 1 - 2 mg / ml peginterferon alpha - 2a , 20 mm acetic acid / sodium acetate ph 6 . 0 , 50 mm sodium chloride ) was spiked with different concentrated excipient stock solutions and at the same time diluted in order to yield final drug product formulations containing 0 . 27 mg / ml peginterferon alpha - 2a , 20 mm acetic acid / sodium acetate ph 6 . 0 , 137 mm nacl , l - methionine at levels indicated below , 0 . 2 mg / ml polysorbate 20 or poloxamer 188 . as a control , the current pegasys drug product market formulation ( 0 . 27 mg / ml peginterferon alpha - 2a , 20 mm acetic acid / sodium acetate ph 6 . 0 , 137 mm nacl , 10 mg / ml benzyl alcohol , 0 . 05 mg / ml polysorbate 80 ) was compounded following the same procedure . after careful homogenization by stirring , all final bulk solutions were sterile filtered using 0 . 22 μm hydrophilic pvdf filters . for stability assessment the solutions were aseptically filled into sterile , pre - siliconized glass syringes ( fill volume : 1 ml ) and closed with sterile rubber stoppers . the samples were stored at 5 ° c . and 25 ° c ., respectively , and analyzed for purity at the time points indicated below using analytical procedures established for the commercial drug product ( size exclusion chromatography and reversed phase hplc ). the analytical methods applied to show the purity of the formulations during storage at different temperatures reveal very similar to almost identical stability properties of the new formulations ( f1 and f2 ) compared to the current benzyl alcohol - containing formulation ( f20 ). f17 which contains neither l - methionine nor benzyl alcohol demonstrates that the presence of an agent with antioxidant properties is required to protect the api from oxidation . f1 : polysorbate 20 containing formulation with 10 mm l - methionine , f2 : poloxamer 188 containing formulation with 10 mm l - methionine , f17 : like f2 but without l - methionine , f20 : current formulation ( 20 mm acetic acid / na - acetate ph 6 . 0 , 137 mm nacl , 10 mg / ml benzyl alcohol , 0 . 05 mg / ml polysorbate 80 ) table 1 below presents data showing the ratio of the purity of pegylated alpha - 2α interferon to monomer content , as measured by size exclusion chromatography , in different formulations over time at 5 ° c . storage . this data is represented in fig1 . table 2 below presents data showing the ratio of the purity of pegylated alpha - 2α interferon to monomer content , as measured by size exclusion chromatography , in different formulations over time at 25 ° c . storage . this data is represented in fig2 . table 3 below presents data showing the ratio of the purity of pegylated alpha - 2α interferon to content of non - oxidized active pharmaceutical ingredient ( api ), as measured by hplc , in different formulations over time at 5 ° c . storage . this data is represented in fig3 . table 4 below presents data showing the ratio of the purity of pegylated alpha - 2α interferon to content of non - oxidized active pharmaceutical ingredient ( api ), as measured by hplc , in different formulations over time at 25 ° c . storage . this data is represented in fig4 .
the present invention provides a stable , isotonic , aqueous solution formulation comprising : 0 . 1 - 0 . 5 mg / ml alpha - type interferon , preferably pegylated alpha - type interferon ; 20 mm acetate buffer system to maintain a ph of 6 . 0 ± 0 . 5 ; 5 - 20 mm l - methionine ; 120 - 150 mm sodium chloride ; 0 . 01 - 0 . 07 percent by weight of a surfactant effective to stabilize the alpha - type interferon , preferably pegylated alpha - type interferon against loss of its activity and an amount of water for injection sufficient to prepare a solution of the above - listed ingredients .