Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/21—Interferons [IFN]
  • A61K38/215—IFN-beta
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
  • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
  • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

• This invention relates, e.g., to pharmaceutical formulations for interferon- ⁇ which comprise a glycine buffer at pH about 2.0 to about 4.0 and which do not contain substantial amounts of human serum albumin or detergent.
• Interferon- ⁇ (“IFN- ⁇ ") is used to treat several medical conditions and is being investigated for a number of others.
• IFN- ⁇ Interferon- ⁇
• recombinantly-produced human IFN- ⁇ is used.
• a genetically engineered version of human IFN- ⁇ in which Ser 17 replaces Cys 17 (“IFN ⁇ -lb"), as described in U.S. Pat. No. 4,588,585 has been approved for treatment of multiple sclerosis.
• the present invention relates, e.g., to low pH (e.g., pH about 2.0 to about 4.0) interferon- ⁇ (IFN- ⁇ ) compositions comprising a glycine buffer.
• the compositions of the invention are stable as liquid formulations and as lyophilizates in the substantial absence of conventional stabilizers (e.g., human serum albumin) and/or solubilizers (e.g., detergents).
• the invention particularly relates to biologically active human IFN- ⁇ , preferably recombinant IFN- ⁇ , including IFN- ⁇ analogs, and most preferably IFN ⁇ -lb, as described in U.S. Pat. No. 4,588,585.
• One aspect of the invention is an IFN- ⁇ composition
• an IFN- ⁇ composition comprising biologically active IFN- ⁇ to which a glycine buffer has been added to achieve a pH of about 2 to about 4, e.g., wherein the buffer further comprises HC1; a composition having a pH of about 2 to about 4, comprising biologically active IFN- ⁇ and a glycine buffer or biologically active IFN- ⁇ and glycine; an IFN- ⁇ composition consisting essentially of biologically active IFN- ⁇ to which a glycine buffer has been added to achieve a pH of about 2 to about 4, e.g.
• the buffer comprises HC1; or a composition having a pH of about 2 to about 4, consisting essentially of biologically active IFN- ⁇ , water and a glycine buffer, or biologically active IFN- ⁇ , water and glycine.
• the water in the compositions of the invention is preferably sterile water which is, e.g., substantially free of pyrogens or trace minerals, most preferably USP grade water for injection (WFI).
• Another aspect of the invention is any of the above IFN- ⁇ compositions, wherein the glycine is in a stabilizing effective amount; wherein the composition is in the form of a pharmaceutical composition, is sterile, or is in a container for parenteral or subcutaneous admmistration (e.g., injection or inhalation); wherein at least 75% of the biological activity of the IFN ⁇ -lb is retained after storage of the composition at 4° C.
• the IFN- ⁇ is unglycosylated and is produced in a bacterial host, e.g., is IFN ⁇ -lb; wherein the composition is substantially free of human serum albumin or detergent and/or is in the substantial absence of glycerol or PEG; wherein the concentration of biologically active IFN- ⁇ is between about 1.0 mg/mL and about 20 mg/mL; and/or wherein the IFN- ⁇ is not in the form of a non-covalently associated aggregate.
• Another aspect of the invention is a lyophilized IFN- ⁇ composition consisting essentially of biologically active IFN- ⁇ and glycine/HCl or biologically active IFN- ⁇ and glycine; or comprising biologically active IFN- ⁇ and glycine.
• the invention also relates to any of the above lyophilized IFN- ⁇ compositions, wherein the IFN- ⁇ is unglycosylated and is produced in a bacterial host, e.g., is IFN ⁇ -lb; or wherein at least 75% of the biological activity of the IFN- ⁇ is recoverable in soluble form after storage of the composition at about 25° C. at least 6 months.
• the invention also relates to a lyophilized IFN- ⁇ composition prepared by lyophilizing a solution having a pH of about 2 to about 4, which consists essentially of biologically active IFN- ⁇ , water (e.g., WFI) and a glycine buffer, to obtain said lyophilized IFN- ⁇ composition; or prepared by lyophilizing a solution having a pH of about 2 to about 4, which comprises biologically active IFN- ⁇ , water (e.g., WFI) and a glycine buffer, to obtain said lyophilized IFN- ⁇ composition.
• a lyophilized IFN- ⁇ composition prepared by lyophilizing a solution having a pH of about 2 to about 4, which comprises biologically active IFN- ⁇ , water (e.g., WFI) and a glycine buffer, to obtain said lyophilized IFN- ⁇ composition.
• Another aspect of the invention is a process for preparing a lyophilized IFN- ⁇ composition, comprising lyophilizing a solution having a pH of about 2 to about 4, consisting essentially of biologically active IFN- ⁇ , water (e.g., WFI) and a glycine buffer, to obtain said
• lyophilized IFN- ⁇ comprising lyophilizing a solution having a pH of about 2 to about 4,
• IFN ⁇ -lb is IFN ⁇ -lb.
• kits comprising a) a container which contains a lyophilized IFN- ⁇ composition as above and b) a container which contains a suitable aqueous solution for reconstituting said composition (e.g., sterile water, preferably sterile, pyrogen-free water, most preferably WFI).
• a suitable aqueous solution for reconstituting said composition e.g., sterile water, preferably sterile, pyrogen-free water, most preferably WFI.
• the composition comprises, or consists essentially of
• 4.0 preferably about 3.0 to about 4.0, more preferably about 3.0 to about 3.5, and most preferably about 3.0, e.g., 2.8 to 3.2, preferably 2.9 to 3.1, provides excellent stability and
• solubility for IFN- ⁇ in liquid formulation or as a lyophilizate in a preferred embodiment, the
• buffer is a glycine buffer which comprises, in addition to glycine, HC1.
• buffers can be used (e.g., aspartic acid or glutamic acid); and many other types of acids can be used to adjust the pH (e.g., phosphoric acid).
• the discussion herein focuses primarily on glycine/HCl buffers. However, one of skill in the art will recognize that this is only exemplary of the many types of buffers which can be used.
• buffers of the invention are that they impart stability and/or solubility to IFN- ⁇ , even in the substantial absence of conventional stabilizers and/or solubilizers, such as
• HSA human serum albumin
• PEG polyethylene glycols
• glycerol polyhydric sugar alcohol
• polyvinylpyrrolidone polyvinylpyrrolidone
• Such stabilizers and solubilizers are disadvantageous in pharmaceutical compositions because they add to the cost of preparation of the compositions, can cause allergic reactions, and may not be compatible with preferred pH conditions for proces- sing, lyophilization and lyophilizate reconstitution.
• Components of the buffers of the instant invention e.g. glycine, are present in the compositions in stabilizing-effective amounts.
• Solubilizers such as SDS which are used to solubilize the inclusion bodies in which heterologous proteins such as IFN- ⁇ are often produced in an aggregated or denatured form by bacteria, must be removed from the heterologous protein during processing, as such solubilizers are toxic and/or denature the biologically active form of the heterologous protein, e.g., by unfolding the native structure of the heterologous protein.
• heterologous proteins produced by bacteria, and particularly IFN- ⁇ produced by bacteria are subject to solubility problems after removal of the solubilizer or SDS.
• An advantage of the present invention is that it provides a stable solution of soluble, biologically active recombinant IFN- ⁇ even in the substantial absence of detergent and/or solubilizer such as, e.g., SDS or Zwit 314.
• Buffers of the invention also minimize the formation of non-covalently associated multimers or aggregates of IFN- ⁇ (i.e., they optimize the formation of non-covalently associated monomers).
• the degree of aggregation can be determined by conventional methods such as, e.g., dynamic light scattering or size exclusion chromatography. Because compositions of the
• stabilizing agents such as, e.g., HSA, the ⁇ -IFN of the
• invention is not aggregated (e.g., complexed) with, e.g., HSA.
• compositions of the invention (either in liquid or lyophilized form) also offer the advantage of being stable under ambient temperature storage conditions. Liquid formulations, therefore, do not need to refrigerate during storage and distribution.
• the invention provides a non-toxic, pharmaceutically acceptable solvent for IFN- ⁇ , particularly unglycosylated IFN- ⁇ , which provides a stable and soluble protein before, during and after lyophilization.
• IFN- ⁇ as used herein encompasses natural human IFN- ⁇ as well as
• Naturally occurring IFN- ⁇ includes that produced by
• fibroblast cells e.g., human foreskin fibroblasts.
• Recombinant human IFN- ⁇ can be produced in any of a variety of host cells, either in a glycosylated form (e.g., in mammalian cells) or in an unglycosylated form (e.g., in bacterial cells).
• Typical host cells include, e.g., mammalian cells, in particular Chinese hamster ovary cells (see, e.g., U.S. Pat. No. 5,376,567).
• the IFN- ⁇ is produced in bacterial cells, preferably E. coli.
• EFN- ⁇ analog is the human recombinant cysteine-replaced mutein, IFN ⁇ -lb, which contains a serine residue in place of the natural unpaired cysteine residue at amino acid 17, as disclosed, e.g., in U.S. Pat. No. 4,588,585.
• the amount of IFN- ⁇ in a liquid formulation that is to be stored as a liquid is preferably about 0.25 mg/mL to about 25.0 mg/mL, more preferably from about 0.5 mg/mL to about 10.0 mg/mL, and most preferably from about 1.0 mg/mL to about 10.0 mg/mL. Within the most preferred range of amounts, the most preferred amount in a liquid formulation that is to be stored
• lyophilized for storage as a lyophilizate is preferably from about 0.25 mg/mL to about 25.0 mg/mL, more preferably from about 0.5 mg/mL to about 10.0 mg/mL, and most preferably from about 1.0 mg/mL to about 10.0 mg/mL. Within the most preferred range of amounts, the most preferred amount in a liquid formulation that is to lyophilized for storage as a lyophilizate is about 5.0 mg/mL.
• CPE cytopathic effect
• the biological activity of the IFN- ⁇ in the formulations of the invention as measured in a CPE-inhibition assay is preferably from about 0.75 X 10 7 IU/mg to about 1.2 X 10 8 IU/mg, more preferably from about 1.0 X 10 7 IU/mg to about 4.5 X 10 7 IU/mg, and most preferably at about 3.0 X 10 7 IU/mg.
• the concentration of glycine in a liquid formulation that is to be stored as a liquid or that is to be lyophilized for storage as a lyophilizate is preferably from about 1 milliMolar (mM) to about 100 mM, more preferably from about 5 mM to about 50 mM, and most preferably at about 20 mM and at about 2 to 5 mM.
• the IFN- ⁇ composition is sufficiently stable such that at least about 50%, preferably at least about 75%, and more preferably at least about 90% of the biological activity is retained after storage of the liquid formulation at 4°C for at least 6 months, preferably at least 9 months, and more preferably at least one year. It is also contemplated that the IFN- ⁇ composition is sufficiently stable such that at least about 50%, preferably at least about 75%, and more preferably at least about 90% of the biological activity is retained after storage of the liquid formulation at ambient temperature ( ⁇ 25 °C) for at least about 6 months, preferably for at least about 9 months, and more preferably for at least about 12 months.
• the IFN- ⁇ composition is sufficiently stable such that at least about 50%, preferably at least about 75%, and more preferably at least about 90% of the biological activity is recoverable in soluble form after storage of the lyophilizate at ambient temperature (approximately 25°C) for at least 2 months, preferably at least 4 months, more preferably at least 6 months and most preferably at least 12 months.
• the IFN- ⁇ composition is sufficiently stable such that at least about 50%, preferably at least about 75%, and more preferably at least about 90% of the biological activity is recoverable in soluble form after storage of the lyophilizate at about 37°C for at least about 6 months, preferably for at least about 9 months, and more preferably for at least about 12 months. It is a further aspect of the invention that the IFN- ⁇ compositions of invention, as either the liquid formulation or as the lyophilizate, are substantially free of detergent and/or solubilizer, e.g., used in the isolation of the protein from the production system.
• the invention particularly relates to such compositions of recombinantly-produced, unglycosylated IFN- ⁇ that are substantially free of detergent and/or solubilizer used in the isolation of protein from the bacterial host.
• substantially free is meant that such IFN- ⁇ compositions have associated with them a
• ppm even more preferably ⁇ 5 ppm, and most preferably ⁇ 2 ppm.
• ppm even more preferably ⁇ 5 ppm, and most preferably ⁇ 2 ppm.
• the amount of detergent is undetectable. For example, for SDS, the lowest amount of SDS which the lowest amount of SDS which is the lowest amount of SDS which is the lowest amount of SDS which is the lowest amount of SDS which is the lowest amount of SDS which is the lowest amount of SDS which is the lowest amount of SDS which is the lowest amount of SDS which is the lowest amount of SDS which is the lowest amount of SDS which is the lowest amount of SDS which is the amount of detergent is undetectable. For example, for SDS, the lowest amount of SDS which
• an "SDS-free" composition is said to comprise ⁇ 25
• compositions which are substantially free of detergent and/or solubilizer are sometimes referred to herein as being in the "substantial absence of detergent and or solubilizer or as having “substantially all” of the detergent and/or solubilizer removed from them.
• the invention also relates to stable lyophilizates of IFN- ⁇ that may be reconstituted in water (e.g., WFI) or other pharmaceutically acceptable aqueous solutions in the absence of substantial amounts of SDS or other detergents/solubilizers such as, for example, Zwit 314, to yield substantially soluble and biologically active IFN- ⁇ .
• WFI water
• SDS detergents/solubilizers
• Zwit 314 Zwit 3144
• lyophilizates that may be reconstituted in parenterally administrable aqueous solutions.
• the solution for reconstitution of the lyophilizate may contain other pharmaceutically acceptable excipients as desired and as are well known in the art.
• the invention also relates to formulations of IFN- ⁇ which are suitable for administration
• the glycine buffered composition can also contain additional conventional pharmaceutically acceptable excipients which provide, for example, improved handling properties.
• Bulking agents such as mannitol or sucrose, for example, can be in amounts which improve the lyophilization characteristics of the IFN- ⁇ /glycine buffered solution.
• the use of mannitol in combination with sucrose is also contemplated.
• the amount of mannitol employed is preferably less than about 50% (w/v), more preferably about 1.0 % to about 5.0% (w/v), and most preferably about 2.0% (w/v).
• the ratio of mannitol/sucrose employed is preferably about 50 parts mannitol to 50 parts sucrose, more preferably about 75 parts mannitol to about 25 parts sucrose, most preferably about 100 parts mannitol to about 0 parts sucrose.
• the total amount of mannitol plus sucrose is preferably about 1.0 % (w/v) to about 5.0% (w/v), more preferably about 2.0% (w/v).
• IFN- ⁇ is bacterially-produced and is recovered from its bacterial host by a process which removes substantially all of the solubilizer, e.g., SDS, used in isolating the IFN- ⁇ from the bacterial inclusion bodies, and which yields a substantially biologi- cally active IFN- ⁇ .
• solubilizer e.g., SDS
• Such methods are taught, e.g., in U.S. Pat. Nos. 4,462,940 and 5,643,566, and in particular in U.S. Pat. No. 5,004,605.
• compositions containing IFN- ⁇ dissolved in a glycine buffered solution, lyophilizates thereof, and lyophilizates reconstituted with water or other conventional pharmaceutically acceptable aqueous media are useful in the same manner as conventional pharmaceutical compositions containing IFN- ⁇ .
• they can be administered to mammals, including humans, for the treatment of various diseases and conditions, e.g., viral diseases, cancer, multiple sclerosis, etc.
• Suitable amounts of IFN- ⁇ and regimens of administration, including routes and frequency of administration for treatment of various diseases and conditions, are well known in the art and can be routinely determined by the skilled practitioner.
• a dosage amount and schedule may be optimized for the individual patient. Optimization of dosage can be determined by monitoring clinical symptoms. Effective dosages are, for example, those which substantially • alleviate the clinical symptoms, and/or slow the progression of, the disease.
• the IFN- ⁇ preparation in accordance with the invention can be formulated in conventional ways standard in the art for administration of protein substances.
• Formulations of the invention are pharmaceutically acceptable for parenteral or non-parenteral delivery; are sterile; and/or are prepared and/or stored in a container (e.g., a vial, ampoule, syringe, etc.) which is suitable for administration to a patient (e.g., is injectable).
• kits comprising: a) a container which contains a lyophilized preparation of IFN- ⁇ according to the invention, and b) a container which contains a suitable sterile aqueous solution for reconstitution of the lyophilizate, e.g., sterile water, which is preferably free of pyrogens of trace minerals.
• a suitable sterile aqueous solution for reconstitution of the lyophilizate e.g., sterile water, which is preferably free of pyrogens of trace minerals.
• the water is USP grade water for injection (WFI).
• WFI USP grade water for injection
• Suitable formulations include solutions or suspensions, or emulsions or solid compositions for reconstitution into injectables or liquid aerosol formulations.
• Acceptable pharmaceutical carriers are those which dissolve the IFN- ⁇ or hold it in suspension and which are not toxic to the patient. Those skilled in the art will know, or be able to ascertain with no more than routine experimentation, particular suitable pharmaceutical carriers for this composition. See, e.g., U.S. Pat. Nos. 4,462,940, 5,643,566 and 5,004,605. Liquid aerosol formulations can be prepared according to the methods employed in, e.g., U.S. Pat. Nos. 5,941,240 and 5,558,085. All materials for the expression, isolation and formulation of IFN- ⁇ and IFN- ⁇ ser ⁇ according to the invention are well known in the art.
• IFN- ⁇ in Escherichia coli is disclosed in Taniguchi et al., Proc. Natl. Acad. Sci. USA (1980), Vol. 77, pp. 5230-5233, and the expression of human IFN- ⁇ in Chinese hamster ovary cells is disclosed in U.S. Pat. No. 5,376,567.
• IFN- ⁇ analogs, such as the human recombinant cysteine-replaced mutein, IFN ⁇ -lb, which contains a serine residue in place of the natural unpaired cysteine residue at amino acid 17, are disclosed, e.g., in U.S. Pat. No. 4,588,585.
• Figure 2 is a graph of the lyophilization cycle for formulations of 0.1 mg/mL IFN ⁇ -lb in 100 mM glycine buffer, pH 3.0 containing either 4% mannitol (w/v) or 4% mannitol (w/v) and 1% sucrose (w/v).
• Figure 15 is an image of an SDS-PAGE analysis of lyophilized and reconstituted samples of 0.1 mg/mL IFN ⁇ - lb in 100 mM glycine buffer, pH 3.0 with either 4% mannitol (Formulation 1) or 4% mannitol and 1% sucrose (Formulation 2), after storage at 50°C for two weeks. Samples are reduced or non-reduced as indicated.
• Figure 16 is a graph of MxA induction results for test samples of 0.1 mg/mL IFN ⁇ -lb in 100 mM glycine buffer, pH 3.0 with either 4% mannitol (Formulation 1) or 4% mannitol and 1% sucrose (Formulation 2), lyophilized and then reconstituted after storage at the indicated temperature for the indicated time.
• biological activity is expressed in International Units per milliliter of solution or IU/mL.
• An international unit is calculated as described in the Research Reference Reagent Note No. 35, published by the National Institute of Health, Bethesda, Maryland, in relation to the HuIFN- ⁇ NIH reference reagent Gb 23-902-531 used as a standard.
• Solubility and Stability as a Function of pH and Additives A solution of purified JJFN ⁇ -lb in 10 mM NaOH, pH 10.8, at a concentration of 0.3-0.5 mg mL (9.6 x 10 6 - 1.6 x 10 7 IU/mL) is used as the starting material.
• the IFN ⁇ -lb is derived from E. coli fermentation of K12/MM294-1 carrying plasmid pSY2501 (ATCC 39517), purified according to the process described in U.S. Patent No. 5,004,605.
• the pH of the starting IFN ⁇ -lb solution is adjusted instantaneously to the desired pH value by the addition of 1/10 volume of a 1 M stock solution of each additive which has previously been titrated to the desired pH value.
• the pH of the resulting IFN- ⁇ solutions is measured to ensure that no significant change in the pH of the additive solution occur as a result of dilution. Additional samples are prepared by adjusting the pH of IFN ⁇ -lb starting solutions to pH 5.0 or pH 6.5 with 1 N acetic acid in the presence or absence of 0.1% SDS.
• IFN ⁇ -lb For samples with less than 30% recovery of IFN ⁇ -lb, a significant amount of visible precipitate forms immediately upon adjustment of the pH of the starting solution.
• the IFN ⁇ -lb is largely insoluble when adjusted to pH values below pH 10.8 and above pH 5.0 unless a solubilizing agent such as SDS is added. Solubility of the IFN- ⁇ can be maintained at pH 5.0 and below after 24 hours of storage at 4°C, depending on the additive with which the pH is adjusted. Both a sodium acetate buffer and an aspartic acid buffer at pH 4.0 solubilize and stabilize IFN- ⁇ significantly. However, a sodium citrate buffer does not maintain the solubility of JJFN- ⁇ . Glycine buffered solution at pH 3.0 gives essentially complete recovery of IFN- ⁇ .
• IFN ⁇ -lb Stability of IFN ⁇ -lb Solutions in 100 mM glycine buffer, pH 3.0 Solutions of purified IFN ⁇ -lb in 100 mM glycine buffer, pH 3 (adjusted with hydrochloric acid) at concentrations of 0.6-1.1 mg/mL (1.9 x 10 7 - 3.5 x 10 7 IU/mL), derived from E. coli fermentation (as described in Example 1 above), are further evaluated for stability. Samples are stored at -70°C, 4°C or 37°C. JJFN ⁇ -lb stability is evaluated by reverse-phase high- pressure liquid chromatography (RP-HPLC) analysis, ELISA analysis, or WISH-CPE bioactivity analysis. The results are presented in Table 2.
• RP-HPLC reverse-phase high- pressure liquid chromatography
• Formulation 1 1.1 mg mL TEN ⁇ -lb in 100 mM NaOAc buffer, pH 5.0.
• Formulation 2 1.1 mg/mL IFN ⁇ -lb in 100 mM NaOAc buffer, pH 5.0 + 0.1% SDS.
• Formulation 3 1.1 mg/mL IFN ⁇ -lb in 100 mM glycine buffer, pH 3.0.
• the IFN ⁇ -lb is derived fromE. coli fermentation as described in Example 1.
• Formulations 1-3 are prepared from G-25 pool of IFN ⁇ -lb by methods similar to those described in U.S. Pat, No. 4,462,940. All three formulations are filtered through a 0.2 ⁇ m filter attached to a syringe. A single filter is used for all formulations to minimize protein loss due to adsorption. The filter is rinsed with water between samples, and the SDS containing formulation is filtered last.
• formulations 1-3 are similar to those obtained by RP-HPLC analysis.
• Formulations 1 and 2 are stable for 1 week at 37°C, while formulation 3 loses approximately 25% of the initial activity.
• the WISH-CPE bioassay has a large standard deviation. Additionally, samples are evaluated on different assay runs because of the limited number of samples that can be analyzed during one run. Formulations 1 and 2 appear to be stable to a freeze-thaw cycle and stable for 1 week at 37°C in Eppendorf tubes or in pumps. For formulation 3, all results are significantly higher than expected and therefore, results for this formulation are difficult to evaluate.
• Formulation 1 is also assayed for bioactivity by measuring down-regulation of TNF expression by activated monocytic cells in culture. These samples are assayed after incubation at 37°C for 1 week and compared to samples stored at 4°C. This assay shows that formulation 1 loses 18% activity after storage at 37°C for 1 week.
• Lyophilized JFN- ⁇ is prepared from a solution of purified recombinant IFN ⁇ -lb in 100 mM glycine buffer, pH 3 (adjusted with hydrochloric acid) at a concentration of 0.1 mg/mL (3.2 x 10 6 IU/mL).
• the IFN ⁇ -lb is derived from E. coli fermentation as described in Example 1 above.
• Tubing vials (5.0 mL) are filled with 1 mL aliquots of the IFN ⁇ -lb solution. After the completion of lyophilization, while still under vacuum, gray butyl rubber stoppers are seated on the vials.
• Lyophilized IFN ⁇ -lb vials are stored at -70°C or at 50°C and reconstituted with 1 mL of water for injection at selected time points in the experiment.
• Reconstituted IFN ⁇ -lb samples are evaluated for IFN ⁇ -lb concentration by RP-HPLC analysis, ELISA analysis, or WISH CPE bioactivity analysis.
• the results of the evaluation of the samples of IFN ⁇ -lb concentration are presented in Table 5 below. Samples incubated at 50°C are also analyzed by SDS- polyacrylamide gel electrophoresis (PAGE).
• Lyophilized IFN ⁇ -lb formulations are prepared from solutions containing 0.1 mg/mL
• IFN ⁇ -lb 100 mM glycine buffer, pH 3.0 and bulking agents consisting of either 4% mannitol (formulation 1) or 4% mannitol and 1% sucrose (formulation 2).
• the IFN ⁇ -lb is derived from E. coli fermentation as described in Example 1 above.
• Tubing vials (5.0 mL) are filled with 1 mL aliquots of the IFN ⁇ -lb solutions. After the completion of lyophilization while still under vacuum, gray butyl rubber stoppers are seated on the vials. Vials are stored at 4°C and 25°C and reconstituted with 1 mL of water for injection at selected time points throughout the experiment. Reconstituted samples are analyzed for IFN ⁇ -lb purity by reverse-phase HPLC (Table 6) and for bioactivity in a WISH CPE assay (Table 7).
• IFN ⁇ -lb Glycine buffer + Mannitol/Sucrose
• Two glycine based, non-HSA containing formulations of IFN ⁇ -lb are tested for stability after lyophilization.
• the two IFN ⁇ -lb formulations are as follows: Formulation 1 : 0.1 mg/mL IFN ⁇ -lb in 100 mM glycine buffer, pH 3.0, with 4% mannitol.
• Formulation 2 0.1 mg/mL IFN ⁇ -lb in 100 mM glycine buffer, pH 3.0, with 4% mannitol and 1% sucrose.
• the IFN ⁇ -lb is derived from E. coli fermentation as described in Example 1.
• the IFN ⁇ -lb is prepared from a G-25 pool of IFN ⁇ -lb (prepared by methods described in Example 3 above) which is further purified over Q-Sepharose (G-25Q) to reduce the level of the carbohydrates.
• G-25Q Q-Sepharose
• Approximately 75 vials of each formulation are filled for lyophihzation and other vials of the IFN ⁇ -lb formulations are filled and stored at -70°C for use as pre-lyophilization control samples.
• West Co. tubing vials (5 mL) are filled with 1.0 mL of formulated solution. Both formulations are lyophilized simultaneously and cycle data from the lyophihzation is shown in Figure 2.
• Samples are frozen to -43°C and held for five hours. Primary drying is conducted at -35°C for 25 hours, followed by -10°C for four hours. Secondary drying is performed at 22°C for 12 hours. Vials are stopped under full vacuum ( ⁇ 50 mTorr) using non- siliconized 20 mM West 4416/50 stoppers.
• WFI water
• Karl Fischer residual moisture is measured at only one time point, using vials which have been stored at -70° C for approximately six months. Karl Fischer analysis is performed using an Aquastar colorimetric titrator, methanol as the extracting solvent, and non-pyridine containing reagents (Coulomat A and C, EM Science). The residual moisture results are similar for the two formulations: 0.63% for formulation 1 and 0.75% for formulation 2 (average of two vials for each formulation).
• Lyophilized samples of formulation 2 are observed to develop a yellow brown color at higher incubation temperatures over time.
• Formulation 2 samples turn yellow between two and eight weeks storage at 37°C. Yellowing is not observed at the 25°C and the 4°C storage conditions for formulation 2.
• Formulation 1 samples remain white under all storage conditions. At all time points, samples of both formulations go into solution immediately ( ⁇ 30 seconds) upon reconstitution. The color of the resulting solutions is clear for all cakes, which are white.
• Formulation 2 samples which are yellow/brown give similarly colored solutions. No turbidity is observed for any samples.
• formulation 1 samples stored at 37°C changes in the reverse phase profile are similar to but more extensive than changes observed in the profile of samples stored at 25°C.
• formulation 2 samples stored at 37°C show significantly increased degradation relative to samples stored at lower temperatures, for which essentially no degradation is detected ( Figures 11 and 12).
• the RP-HPLC analysis demonstrates that degradation of IFN ⁇ -lb in formulation 1 results in broadening of the main IFN ⁇ -lb peak and an increase in the amount of late-eluting peaks, which apparently correspond to late-eluting peaks that are present in the prelyophilization samples in low amounts. Therefore, the degradation path(s), as detected by RP- HPLC, appears to be similar over the examined temperature range, and the amount of degradation increases with increasing storage time and temperature. In contrast, there is a significantly increased degradation of formulation 2 at 37°C, relative to lower storage temperatures. Extensive degradation is detected as significant broadening of the main IFN ⁇ -lb peak.
• late-eluting material is not resolved as the peaks present at low levels in the prelyophilization samples.
• the degradation pathways may be different for samples stored at elevated temperatures than for samples stored at lower temperatures.
• Reconstituted samples at most storage time/temperature points are analyzed by ELISA and show highly variable results (Table 10). There appears to be a significant amount of ELISA activity remaining in samples of both formulations after storage for 25 weeks at any of the studied temperatures.
• Reconstituted samples are also analyzed by SDS-PAGE.
• the only apparent change observed in IFN ⁇ -lb is the shift to slightly higher molecular weight of the IFN ⁇ -lb band for formulation 2 samples that have been stored at 37°C for 25 weeks. This change is observed in both reduced and non-reduced samples.
• glycine buffer means glycine to which sufficient acid has been added to achieve the stated pH. Also inherent is that ingredients typically found in glycine buffers can be used as long as not incompatible with the use of IFN- ⁇ . Ingredients may include inorganic salts such as, e.g., Mg, Zn, K, Na, etc., as long as no adverse effects on patients are caused by the salt.

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