MXPA99007663A - Stabilized compositions of prote - Google Patents

Abstract

The present invention relates to stabilized protein compositions, methods for preparing said compositions, dosage forms for administering the same to mammals, and methods for preventing or treating infections in mammals, administering said protein compositions thereto; stabilized protein compositions of the present invention contain therapeutically effective amounts of G-CSF, such as bovine G-CSF, in combination with a stabilizing pH regulator, such as HEPES, TES or TRICINE, to treat and prevent infections, including mastitis, in the win

Description

> ** of 1987 (Hasegawa et al.), Relates to a pharmaceutical composition for treating viral infections containing interferon, a tripolyhydric or higher sugar alcohol, an organic regulatory solution and a pharmaceutical carrier or diluent, wherein the composition has a pH of approximately 3 to 6. All the aforementioned references are ,? incorporated herein by reference in their entirety An example of a therapeutically effective class of protein is that of granulocyte colony stimulation factors (G-CFS). The granulocyte colony stimulation factor (G-CSF) is one of several 10 glycoprotein growth factors known as »Colony stimulation. Such colony stimulation factors support the J. proliferation of hematopoietic progenitor cells and stimulate the * l * proliferation of specific precursor cells of the bone marrow and its differentiation in granulocytes. In addition, G-CSF allows the stimulation of neutrophil granulocyte colony formation and induces differentiation. * * * * s terminal murine myelomonocytic murine cells in vitro. It has also been shown that G-CSF stimulates the functional activities of neutrophil X X which results in increased microbicidal activity. The G-CSF < and has a known amino acid sequence of 174 amino acids. * », Su" * 20 The recombinant forms of the CSF and the -, - * -> G-CSF have been prepared The cloning and expression of DNA encoding human G-CSF is known (Nagata, S and others, Nature, 319, 415-418 (1986). The documents lt WO-A-8604606 and WO-A-8604506 describe a gene coding for G-CSF.

*,.* Be human. The Patent E.U.A. No. 5,606,024 issued February 25, 1997 (Boone et al.) And Patent E.U.A. No. 5,472,857 issued December 5, 1995, describes the DNA sequence encoding the canine granulocyte colony stimulation factor (cG-CSF) as well as a method to treat or prevent infections in canine or feline animals by administering amounts ? .1 effective human and canine G-CSF to such animals. The U.S. Patent: A. No. 4,810,643 issued March 7, 1989 (Souza), describes human G-CSF type polypeptides. European Patent Application No. 719 860 published on 3 July 1, 1996, describes the amino acid sequence of the stimulation factor of naturally occurring bovine granulocyte colony. (bG-CSF), the DNA sequence encoded for bG-CSF and a method • - • ** to treat or prevent mastitis in an animal by administering to the animal an effective amount of G-CSF WO-A-8702060 describes a human G-CSF-like polypeptide, the sequences encoding the same Y methods to produce them. The patent E.U.A. No. 4,833,127 issued on 23 May 4, 1989 (Ono et al.), Describes a novel biologically active human granulocyte colony stimulation factor. European Patent Application No. 612 846, published on August 31, 1994, discloses certain analogs of G-CSF and compositions containing such analogs. All references mentioned above are incorporated herein by reference in their entirety. Granulocyte colony stimulation factors are useful as anti-infective agents that increase the animal's immune competence rather than being directed to a specific microbial target necessary for its growth or virulence. There are a few different commercially available agents used in veterinary medicine that are aimed at giving non-specific immune responses which leads to increased resistance in terms of microbial infection. The available control measures are limited to conventional antimicrobials and a limited number of biological products. The economic losses associated with milk milking periods in cattle limit the utility of conventional antimicrobials. Current vaccines are directed to a number The limited number of species and the field efficacy of these agents vary widely. The most successful vaccines, (E. Coli J5) are limited in their use worldwide due to concerns regarding safety associated with endotoxin contamination. Mastitis is a major problem of disease that affects dairy producers worldwide. The economic losses in the United States associated with mastitis exceed one billion dollars annually. These losses are associated with mortalities, milk waste, acute and chronic decreases in milk production, increased early selection and veterinary labor costs, and of drugs. Periparturient milk producing cows exhibit impaired immune response capacity (neutrophil function) which increases their susceptibility to bacterial infections of the mammary gland. The impact of this increased susceptibility is exemplified by the fact that approximately 40% of new intramammary clinical infections occur within the first 2 weeks after delivery. Mastitis is associated with a wide variety of bacterial pathogens that include both Gram positive and Gram 5 negative organisms. Some of the known pathogenic microorganisms that cause mastitis are Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, Streptococcus dysgalactiae, Aerobacter aerogenes, Klebsiella pneumoniae and Pseudomonas aeruginosa. These pathogens enter the udder through the teat canal and produce inflammation of the milk producing tissue causing scar tissue formation, which can result in a permanent loss of milk production capacity. The various forms of mastitis include: udder infection, chronic mastitis, clinical mastitis and subclinical mastitis. Current antimicrobial therapies and vaccines have a 15 number of deficiencies that limit their usefulness in lactating cows. It has been found that antibiotic therapy to control mastitis is insufficient. There is a need for a biotherapeutic agent that is useful for restoring normal immune competence that results in decreased # «2T» of the incidence and severity of mastitis. f «* 2Ít« f * * "20 Bovine respiratory disease, also known as i i. Disease of boarding, is another common disease that affects cattle. Bovine respiratory disease affects livestock after boarding either in feedlots or in grazing and results from a variety of adverse situations affecting livestock including weaning, castration, dehorning, fasting, overcrowding, exposure to infectious agents, changes in diet and changes in temperature, in combination with infection by any of several known pathogens . 5 Pasteurella haemolytica is one of such common pathogens that results in damage to the respiratory system of livestock It is also known that various additional infectious diseases, including various reproductive diseases, affect humans, pigs, cattle, dogs, cats, horses, goats and sheep. . An example of a disease as such, which occurs in cattle, is metritis. There is a need for a stable protein composition that remains therapeutically effective for extended periods in vivo. In addition, there is a need for protein formulations that provide - - 4 15 storage and shelf life prolonged in vitro. i% ? d: GO BRIEF DESCRIPTION OF THE INVENTION The present invention relates to a stabilized protein composition comprising a protein and a buffer stabilizing solution whose composition makes it possible to maintain therapeutic levels of said protein for a prolonged period. ; i. * Specific embodiments of the invention include a stabilized protein composition whose composition is at a physiological pH. Other specific embodiments of the invention include a stabilized protein composition whose composition is at a physiological temperature. Other specific embodiments of the invention include a stabilized protein composition wherein the stabilizing buffer is selected from the group consisting of: HEPES, TES, and TRICINE. Even other specific embodiments of the invention include a stabilized protein composition wherein the prolonged period is at least three days. Even other specific embodiments of the invention include a protein stabilized composition wherein the protein is selected from the group consisting of: colony stimulation factors, somatotropins, interleukins, interferons, cytokines, antibodies and antigens. More specific embodiments of the invention include a stabilized protein composition wherein the protein is selected from the group consisting of: human G-CSF, bovine G-CSF and canine G-CSF. r * The most specific embodiments of the invention include a stabilized protein composition wherein the protein is G-CSF and en-3 * t where G-CSF is present in a concentration in the range of 0.01 to 5 mg / ml. Other specific embodiments of the invention include a stabilized protein composition wherein the protein is G-CSF and wherein the stabilizing buffer is selected from the group ! • * consists of: HEPES, TES and TRICINE. Other more specific embodiments of the invention include a stabilized protein composition wherein the protein is G-CSF and in "* s" ^ wherein the stabilizing buffer is present in a concentration ranging from 0.05 M to about 2M. Still other specific embodiments of the invention include a stabilized protein composition wherein the protein is G-CSF and wherein the composition is at a physiological pH. Even other specific embodiments of the invention include a stabilized protein composition wherein the protein is G-CSF and wherein the composition is at a physiological temperature. More specific embodiments of the invention include a stabilized protein composition wherein the protein is bovine G-CSF. Other specific embodiments of the invention include a stabilized protein composition wherein the protein is bovine G-CSF and wherein bG-CSF is present in a concentration in the range of 0.01 to 5 mg / ml.

Other specific embodiments of the invention include a stabilized protein composition wherein the protein is bovine G-CSF and wherein the stabilizing buffer is selected from the group consisting of: HEPES, TES and TRICINE. Even in other specific embodiments of the invention a stabilized protein composition is included wherein the protein is bovine G-CSF and wherein the stabilizing buffer is present at a concentration ranging from 0.05 M to about 2M. Even other specific embodiments of the invention include a stabilized protein composition wherein the protein is bovine G-CSF and wherein the composition is at a physiological pH. Still other specific embodiments of the invention include a stabilized protein composition wherein the protein is bovine G-CSF and wherein the composition is at a physiological temperature. Preferably, the stabilized protein composition of the invention is a composition composed of bovine G-CSF in HEPES buffer. More preferably, the HEPES buffer is in a concentration ranging from 0.05M to about 2M. Such formulations of bovine G-CSF are preferably at such a physiological pH as 7.5. In addition, such preferred bovine G-CSF formulations can maintain, for a prolonged period, from at least 3 days to 7 days or longer,: therapeutic levels of bovine G-CSF.

In addition, the stabilized protein composition of the invention is a composition consisting of bovine G-CSF in buffer solution of HEPES whose composition allows to provide a shelf life and prolonged storage. Preferably the HEPES buffer is in a concentration ranging from 0.05M to about 2M. More preferably, said contents of the compositions are maintained at a pH of about 4.0 to 7.5, preferably 4.0, and at a temperature of less than about 40 ° C and preferably about 4 ° C. The shelf life and long storage are in the range from 3 weeks to about 18 months, and preferably are in the range of 6 weeks to about 1 year. The present invention further relates to a pharmaceutically acceptable dosage form of a stabilized protein composition for parenteral administration to a mammal, consisting of a protein and a pharmaceutically acceptable stabilizing buffer, whose composition makes it possible to maintain therapeutic levels of such a protein. for a prolonged period, wherein the protein is present in an amount sufficient to provide protection to the mammal for a prolonged period. Specific embodiments of the invention include a pharmaceutically acceptable dosage form wherein the dosage form further comprises a component that is selected from the group consisting of viscosity modifiers and surfactants.

The present invention also relates to a method for preparing a pharmaceutically acceptable dosage form of a stabilized protein composition for parenteral administration to a mammal, comprising the step of combining a protein and a stabilizing buffer, whose stabilized composition of protein allows to maintain therapeutic levels of such protein for a prolonged period, wherein the protein is present in an amount sufficient to provide protection to a mammal for at least 3 days. The present invention also relates to a method of treating or preventing infections in mammals which comprises administering to the mammal a stabilized protein composition comprising administering to the mammal a therapeutically effective amount of a stabilized protein composition, wherein the composition The stabilized protein consists of a protein and a stabilizing buffer, whose composition allows maintaining therapeutic levels of such a protein for a prolonged period. Specific embodiments of the invention include a method such as treatment or prevention of infections in mammals wherein the protein is G-CSF. The present invention also relates to a method of treatment for prevention of mastitis, metritis or bovine respiratory disease in cattle, which comprises administering to the mammal a stabilized G-CSF composition comprising administering to the mammal a therapeutically effective amount of a stabilized composition of G-CSF, wherein the stabilized composition of G-CSF consists of G-CSF and a stabilizing buffer, whose composition allows to maintain therapeutic levels of such a protein for a prolonged period. The present invention also relates to a method for maintaining therapeutic levels of a protein in a mammal for a prolonged period, which comprises administering to the mammal a stabilized protein composition, wherein the stabilized protein composition comprises a protein and a buffer solution. stabilization, whose composition allows to maintain therapeutic levels of such protein for a prolonged period. Specific embodiments of the invention include a method as such for maintaining therapeutic levels of a protein in a mammal for a prolonged period, wherein the stabilizing buffer is selected from the group consisting of: HEPES, TES and TRICINE. Other specific embodiments of the invention include a method as such for maintaining therapeutic levels of a protein in a mammal for a prolonged period, wherein the prolonged period is at least 3 days. Other specific embodiments of the invention include a method as such for maintaining therapeutic levels of a protein in a mammal for a prolonged period, wherein the protein is selected from the group consisting of: colony stimulation factors, somatotropins, cytokines, antibodies and antigens. Specific examples of cytokines include interleukins, such as interleukins 1-18, and interferons such as interferons, β and β. The more specific embodiments of the invention include a method as such for maintaining therapeutic levels of a protein in a mammal for a prolonged period, wherein the protein is a colony stimulating factor. Even in other specific embodiments of the invention there is included a method as such for maintaining therapeutic levels of a protein in a mammal for a prolonged period, wherein the protein is selected from the group consisting of: human G-CSF, bovine G-CSF and G-canine CSF. Still other specific embodiments of the invention include a method as such for maintaining therapeutic levels of G-CSF in a mammal for a prolonged period, wherein the G-CSF is present in a concentration ranging from 0.01 to 5 mg / ml. Even other specific embodiments of the invention include a method as such for maintaining therapeutic levels of G-CSF in a mammal for a prolonged period, wherein the stabilizing buffer is selected from the group consisting of: HEPES, TES and TRICINE. Still other specific embodiments of the invention include a method as such to maintain therapeutic levels of G-CSF in a mammal for a prolonged period wherein the stabilizing buffer is present in a concentration ranging from 0.05 M to about 2M. Even in other specific embodiments of the invention they include a method as such for maintaining therapeutic levels of G-CSF in a mammal for a prolonged period, wherein the G-CSF is bovine G-CSF. Even other specific embodiments of the invention include a method as such for maintaining therapeutic levels of bG-CSF in a mammal for a prolonged period, wherein bG-CSF is present in a concentration ranging from 0.01 to 5 mg / mol. Even other specific embodiments of the invention include a method as such for maintaining therapeutic levels of bG-CSF in a mammal for a prolonged period, wherein the stabilizing buffer is selected from the group consisting of: HEPES, TES and TRICINE. Still other specific embodiments of the invention include a method as such to maintain therapeutic levels of bG-CSF in a mammal for a prolonged period, wherein the stabilizing buffer is present in a concentration ranging from 0.05 M to about 2 M The present invention further relates to a kit for administering to the mammal a stabilized protein composition consisting of a first container having a therapeutically effective amount of a protein and a second container having a pharmaceutically acceptable stabilizing buffer solution, wherein The therapeutically effective amount of the protein of the first container when combined with the pharmaceutically acceptable stabilizing buffer solution of the second container allows maintaining therapeutic levels of such a protein in the mammal for a prolonged period. Specific embodiments of the invention include a kit wherein the protein is present in an amount sufficient to provide Yes. Protection of a mammal for at least three days. A preferred composition of the invention is a stabilized protein composition consisting of bovine G-CSF and HEPES buffer, the composition of which allows therapeutic levels of bovine G-CSF to be maintained in a mammal, in vivo, for at least three days, wherein the composition is at a pH of about 7.5 and wherein the The composition is at a temperature close to the physiological temperature or 40 ° C. A composition as such is particularly useful where the mammal is a cow. More particularly, the HEPES regulatory solution is present at a concentration ranging from 0.05M to about 2M. This is particularly preferable where the regulatory solution of HEPES is present at a concentration about 1 M. Preferably, the bovine G-CSF is present at a concentration in the range of 0.1 to 5 mg / mL. More preferably, the concentration of bG-CSF is about 0.1 mg / mL.

Preferably, the invention also relates to a stabilized protein composition consisting of G-CSF and HEPES buffer, whose composition allows to provide a prolonged shelf life in the range of 3 weeks approximately 18 months. This is particularly preferred where the HEPES buffer is at a concentration ranging from 0.05 M to about 2M. It is also preferred where the composition is at a pH of about 7.5 and where the temperature of the composition is less than 40 ° C, preferably about 4 ° C. It is also particularly preferred where the prolonged shelf life is in the range from 6 months to about 1 year. Alternatively, such a stabilized composition that allows a prolonged shelf life can be maintained at a composition temperature of about 40 ° C.

DESCRIPTION OF THE FIGURES Figure 1 shows the stability (% recovery) of bG-CSF solutions of 0.1 mg / ml at pH 7.5, as a function of time, under storage conditions of 40 ° C in 0.1 M HEPES buffer concentrations , 1 M and 2M. Figure 2 shows the stability (% recovery) of bG-CSF solutions of 0.1 mg / ml at pH 7.5, as a function of time, under storage conditions of 40 ° C in 0.1 M TES buffer concentrations. , 1 M and 2M. Figure 3 shows the stability (% recovery) of bG-CSF solutions of 0.1 mg / ml at pH 7.5, as a function of time, under storage conditions of 40 ° C in TRICINE buffer concentrations of 0.1 M , 1M and 2M. Figure 4 shows the stability (% recovery) of bG-CSF solutions of 2 mg / ml at pH 7.5, as a function of time, under storage conditions of 40 ° C in 0.1 M HEPES buffer concentrations , 1 M and 2M. Figure 5 shows the stability (% recovery) of bG-CSF solutions of 2 mg / ml at pH 7.5, as a function of time, under storage conditions of 40 ° C in 0.1 M TES buffer concentrations. , 1 M and 2M. Figure 6 shows the stability (% recovery) of bG-CSF solutions of 2 mg / ml at pH 7.5, as a function of time, under storage conditions of 40 ° C in concentrations of 0.1 M TRICINE buffer. , 1 M and 2M. Figure 7 shows PMN counts of peripheral blood (expressed as% control, value 0 hour) for cattle treated with bG-CSF formulated in water, 1 M HEPES buffer, 1 M TES buffer and 1M TRICINE buffer.

Figure 8 shows the stability of bG-CSF (concentration in mg / mT) as a function of time in Neupogen® buffer solution (control, pH 4.0), HEPES buffer at pH 7.4, PBS at pH 7.0, Hanks buffer at pH 8.5 and bicarbonate buffer to pH 8.2. Figure 9 shows the stability of bG-CSF (concentration in 1 mg / ml) as a function of time in HEPES buffer at 1000mM, 500mM, 100mM, 50mM, and 20mM at 40 ° C.

«? Figure 10 shows two thermograms (kcal / moles / degrees) versus temperature (° C) for two solutions of bG-CSF. The upper thermogram, with a maximum temperature of 47 ° C is for bG-CSF formulated in PBS at pH 7.5, and the lower thermogram, with a maximum temperature of 59 ° C, is for bG-CSF formulated in 1 M HEPES at pH 7.5. Figure 1 1 shows a plot of% PMN (neutrophil) in 15 cattle as a function of time, for three formulations: bG-CSF in water (as a control), bG-CSF in 1 M HEPES and bG-CSF in HEPES 1 M + 10% polaxamer. Figure 12 shows the solubility of bG-CSF in 1 M HEPES buffer at pH 7.5 as measured by absorbance at 310 nm '• * 20 versus concentration of bG-CSF (mg / ml). Figure 13 shows the stability (% initial concentration) of bovine G-CSF in 1 M HEPES buffer and in PBS at 40 ° C.

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Figure 14 shows the stability (% initial concentration) # of human G-CSF in buffer solution of 1 M HEPES and in PBS at 40 ° C. Figure 15 compares the stability (% initial concentration) of human G-CSF and bovine G-CSF in buffer solution of 1 M HEPES at pH 7.5. Figure 16 shows the percentage of initial concentration of, X bG-CSF in 1 M HEPES buffer at pH 4.0 versus time (days) at & 40 ° C. Figure 17 shows the percentage of initial concentration of 10 bG-CSF in 1 M HEPES buffer at pH 7.5 versus time (days) at 40 ° C. Figure 18 shows the percentage of initial concentration of bG-CSF in 1 M TES buffer at pH 4.0 versus time (days) at 40 ° C. 15 Figure 19 shows the percentage of initial concentration of bG-CSF in 1 M TES buffer at pH 7.5 versus time (days) at f, 40 ° C. Figure 20 shows the results of CLAR RP for the percentage of initial concentration of bG-CSF for samples stored at 20 5 ° C versus time (weeks). Figure 21 shows the results of CLAR SE for the percentage of initial concentration of bG-CSF for samples stored at 5 ° C versus time (weeks).

Figure 22 shows the results of CLAR RP for the percentage of initial concentration of bG-CSF for samples stored at 30 ° C versus time (weeks). Figure 23 shows the results of CLAR SE for the 5 percent initial concentration of bG-CSF for samples stored at 30 ° C versus time (weeks). Figure 24 shows the results of CLAR RP for the percentage of initial concentration of bG-CSF for samples stored at 40 ° C. Figure 25 shows the results of CLAR SE for the percentage of initial concentration of bG-CSF for samples stored at 40 ° C versus time (weeks). Figure 26 is the CD spectrum (circular dichroism) of bG-rt CSF. Figure 27 is a graph of the molar ellipticity at a wavelength of 222 nm as a function of temperature. Figure 28 shows the percentage of initial concentration of bG-CSF in various concentrations of HEPES buffer at pH 7.5 at 40 ° C versus time (days). Figure 29 is a graph of WBC versus time after injection (in hours) for bg-CSF formulated in 1 M HEPES regulatory solution versus a control formulation.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to stabilized protein compositions based on the surprising discovery that proteins, and in particular, proteins useful for treating infections in mammals, such as humans, dogs, cats, goats, sheep, horses and pigs, can be stabilized by the addition of a stabilizing buffer, such as HEPES, TES and TRICINE to the protein in such a way that the stabilized protein composition allows to maintain a prolonged period of protein activity both in vivo and in vitro. With respect to in vivo activity, the stabilized protein compositions of the present invention can maintain therapeutically effective levels of such proteins in a mammal for a prolonged period. In particular, the present invention is a formulation of sustained release (sustained activity) of bG-CSF in a solution -, stabilization regulator, such as HEPES or TES, which provides a prolonged therapeutic drug activity. It is known that bG-CSF is denatured at temperatures around 40 ° C and is unstable at neutral pH. This is a concern since the physiological pH is close to neutrality and the body temperature of a cow is about 40 ° C. The proteins of the stabilized compositions of the present invention may be naturally occurring proteins, isolated or purified proteins, or proteins produced recombinantly.

Also included within the invention are all proteins that have been chemically modified by chemical modification of proteins? f r such as oxidation of methionine, alkylation S of cysteine and addition of disulfide with beta-mercaptoethanol, alkylation of amino groups of lysine etc. A preferred protein to be used in the stabilized protein compositions of the present invention is G-CSF, and more preferred is the bG-CSF protein. - With "G-CSF" it is meant colony stimulation factor of • «granulocyte, including the granulocyte colony stimulation factor in Its natural form as well as all variants and mutants thereof, including for example, recombinant variants having one or more deletions, substitutions and / or amino acid additions. Such variants and mutants maintain all or sufficient biological activity to provide a therapeutic benefit to a mammal. The G-CSF in its natural form is a "" •? 15 glycoprotein consisting of a protein that has 174 amino acids, and a form that has 3 additional amino acids. Both forms have five cysteine residues, four of them forming two disulfide bonds and one of them in free form. Other examples of appropriate proteins to be used in The stabilized protein compositions of the present invention include, for example, activins, adhesion molecules such as L-selectin, CD-18 and ICAM-1, chemokines, chemotactic factors, erythropoietin, growth, inhibins, insulin, interferons such as α, β and β; interleukins * - "- i > & 25 such as interleukins 1-18, leptin, inflammatory macrophage proteins, macrophage migration inhibiting factor, macrophage stimulating protein, neurotrophins, neutrophil inhibiting factor, oncostatins, somatostatins, somatotrophins (all species), such as porcine , bovine or human, stem cell factors, tumor necrosis factors, thrombopoietins and soluble receptors associated with cell for all. proteins mentioned above and for any and all other proteins that when administered to a mammal allow to provide a beneficial or therapeutic result. Examples of particular proteins that can be The proteins used in the stabilized protein compositions of the present invention are shown in Table 1. Other proteins that can be used in the stabilized protein compositions of the present invention include those described in R &D Systems Catalog. , 614 McKinley Place NE, Minneapolis MN 55413, USA, incorporated herein invention for reference. í TABLE 1 TABLE 1 (CONTINUED) Proteins with potential therapeutic benefit Fibroblast Growth Factor 8 (FGF-8) Fibroblast Growth Factor 8b (FGF-8b) Fibroblast Growth Factor 8C (FGF-8c) Fibroblast Growth Factor 9 (FGF-9) Factor Fibroblast Growth Acid (FGF acid) Basic Fibroblast Growth Factor (FGF basic) Fibronectin (FN) Fit-1 Ligand Fit-3 Fractalcin Neurotropic Factor Derivative of the Cell Line of the Glia (GDNF) Glycoprotein 130 (gp 130) Chemotactic protein of Granulocyte (GCP-2) Granulocyte Colony Stimulation Factor (G-CSF) Granulocyte Colony Stimulation Factor Receptor (G-CSF R) Granulocyte Macrophage Colony Stimulation Factor (GM-CSF) Related Protein Growth (GRO) Growth-related alpha protein (GROa) Growth-related beta protein (GROß) Growth-related gamma protein (GRO?) Hemophilic CC chemokine I (HCC-1) Heparin-binding Epidermal Growth Factor (HB) -EGF) Hepatocyte Growth Factor (HGF) Heregulin alfa (HRG-a) Heregulin beta 1 (HRG-ß1) -309 Insulin-like Growth Factor (IGF-1) Interferon gamma (IFN.?) Interleukin Receptor Antagonist 1 (IL-1 ra) Interleukin 1 1 receptor (IL-1 1 R) Interleukin 12 p70 (IL-12 p70) Interleukin 13 (1L-13) Interleukin 16 (IL-16) Interleukin 2 alpha receptor (I L -2 Ra) Interleukin 2 beta receptor (IL-Rß) Interleukin 3 (IL-3) Recept of Interleukin 4 (IL-4 R) Interleukin 5 (IL-5) * TABLE 1 (CONTINUED) Proteins with potential therapeutic benefit Interleukin I beta (IL-lß) Interleukin I receptor type II (IL-1 Rll) Mac-1 alpha chain Macrophage Colony Stimulation Factor (M-CSF) - Stimulation Factor Receptor Macrophage Colony (M-CSF R) Inflammatory Macrophage Protein 1 gamma (MlP-1?) Inflammatory Macrophage Protein 2 (MlP-2) Inflammatory Macrophage Protein 3 alpha (MIP-3a) Inflammatory Macrophage Protein 3 beta (MIP) -3ß) Inflammatory Macrophage Alpha I protein (MlP-1a) Inflammatory Macrophage beta I protein (MIP-1 ß) Macrophage Migration Inhibitor Factor (MIF) Macrophage Stimulation Protein (MSP) Chemokine Derived from Macrophage (MDC / DC-CK1) MARC / MCP-3 Midcine (MK) MIG Protein 1 / Monocyte Chemootracic MCAF (MCP-1) Protein 2 Monocyte Chemoattractant (MCP-2) Protein 3 Chemoattractant Monocyte (MCP-3) Protein 4 Chemoattractant Monocyte (MCP-4) Protein 5 Quimiotá Monocyte clique (MCP-5) Nervous Cell Adhesion Molecule (NCAM)) Neurotrophin 3 (NT-3) Neurotrophin 4 (NT-4) With potential therapeutic benefit Oncostatin M (OSM) P-Selectin (CD62P) Growth Factor of Placenta (PIGF) Placenta Growth Factor 2 (PIGF-2) Glutathione Plasma Selenium Peroxidases. Platelet-derived GPIIb / GPIIIa (CD41 a) Platelet-derived Endothelial Cell Growth Factor (PD-ECGF) Platelet-derived Growth Factor (PDGF) Chain A Growth Plate-derived Growth Factor (PDGF A Chain) Growth Factor AA Platelet Derivative (PDGF-AA) Platelet Derived AB Growth Factor (PDGF-AB) Platelet Derived Growth Factor B Chain (PDGF B Chain) TABLE 1 (CONTINUED) Proteins with potential therapeutic benefit Platelet-derived Growth Factor BB (PBGF-BB) Alpha receptor Platelet-derived Growth Factor (PDGF Ra) Beta receptor-derived Platelet Growth Factor (PDGF Rβ) Pleiotropin (PTN) Factor / SDF -1 Pre-B Cell Growth Stimulation (PBSF) RANTES Leukocyte Protease Secretion Inhibitor (SLPI) Stem Cell Factor Receptor (SCF R) Stem Cell Factor (SCF) Factor 1 beta / PBSF Cell Derivative of Stromal (SDF-lß) Factor 1 / PBSF Stromal Cell Derivative (SDF-1) Alpha / PBSF Factor Stromal Alpha / PBSF Derivative (SDF-la) Thrombopoietin (Tpo) Chemokine of Thymus and Regulated Activation (SDF-1) TARC) 10 Chemokine expressed in Thymus (TECK) Transforming Growth Alpha Factor (TGF-a) Transforming Growth Beta Factor (TGF-β) Transforming Growth Beta 1.2 (TGF-β1.2) Transforming Growth Beta 2 Factor (T GF-ß2) Protein I that binds to Transforming Growth Beta-Factor (TGF-ß bpl) Transforming Growth Beta I Factor (TGF-ß1) Transforming Growth Factor Beta-II (TGF-ßRII) receptor Type II Receptor Factor beta Transformant Growth (TGF-ß RUI) 15 ***** Transforming Growth Factor / Beta 5 (YGF-ß5) Transforming Growth Beta 3 Factor (TGF-ß3) TrkB Tumor Necrosis Alpha Factor (TNF-α) ) Tumor Necrosis Beta Factor (TNF-ß) Type I Tumor Necrosis Factor Receptor (TNF Rl) Type II Tumor Necrosis Factor Receptor (TNF Rll) Vascular Cell Adhesion Molecule I (VCAM-1) Growth Factor Vascular Endothelial (VEGF) - 20 * • se ^ * í Preferred proteins are those that are useful for the 4't t • \ * k »* - treatment or prevention of infections in mammals such as humans, dogs, cows, pigs, goats, sheep, horses and cats. Such infections can be bacterial infections or protozoal infections or they can be caused by viruses. As used herein, unless otherwise indicated, the term "infection (s)" includes bacterial, protozoan, fungal, and viral infections that occur in mammals, as well as disorders related to such infections that can be treated or prevented by administering the stabilized protein compositions of the present invention. Infectious diseases that can be treated using the stabilized protein compositions of the present invention include, but are not limited to, infectious livestock diseases such as, for example, bovine mastitis, associated with but not limited to Staphylococcus aureus, Escherichia coli, Streptococcus uberis , Streptococcus dysgalactia, Streptococcus, agalactiae, Klebsiella sp. Corynebacterium sp.; bovine respiratory disease, associated with but not limited to infectious bovine rhinotracheitis virus (IBR), parainfluenza virus (PI3), bovine viral diarrhea virus (BVD), Pasteurella haemolitica, Pasteurella multocida and Haemophilus somnus; reproductive diseases such as metritis; and bovine diarrhea associated with but not limited to E. coli and Eimeria sp. Other examples of infectious diseases that can be treated using the stabilized protein compositions of the present invention include, but are not limited to, infectious diseases of dogs such as bioderma and respiratory disease in dogs, also known as Kennel's cough. The stabilized protein compositions of the present invention can be used to provide therapeutic benefits different those for treatment or prevention of infections. An example of a therapeutic benefit or effect different from the treatment or prevention of infections is the administration of human G-CSF recombinant to dogs and cats to alleviate myelosuppression induced by chemotherapy and to allow more aggressive treatment protocols A t 10 cancer. As used herein, the word "stabilization", except when indicated otherwise, refers to sustained therapeutic levels of the protein, for a prolonged period. Such sustained therapeutic levels of the protein may occur, either after administration to a mammal, or m vitro, before being used or during storage of the stabilized protein composition of the invention. The stability of the protein compositions of the invention can be terminated, for example by the percentage of initial concentration versus time, using the methods described herein: After administration to a mammal, the The stabilized compositions of the present invention provide sustained therapeutic levels of the protein, so that the protein can provide its beneficial or therapeutic effects over a prolonged period As used herein, and unless otherwise indicated otherwise, the term "extended period" refers to that period in which the therapeutic levels of the protein are maintained, either after administration to a mammal or, alternatively in vitro, before use or during storage of the protein. The stabilized composition of the protein of the invention A prolonged period of therapeutic protein levels provides a beneficial effect or therapeutic in the mammal for a longer period than would be possible by administering the same protein to the mammal without the presence of the stabilizing buffer, as compared to for example a control solution of the protein in water or PBS. Alternatively, under in vitro storage conditions, the prolonged period of therapeutic protein levels provides for increased stability of the protein for a longer period than would be possible by storing the same protein under conditions without the presence of the buffer. of stabilization so that when compared to for example a control solution of the protein in water or PBS. Preferably, the prolonged period is at least three days. More preferable, the prolonged period is around seven days or longer. As used herein, and unless otherwise indicated, the term "therapeutic levels" refers to the amount of a protein that provides therapeutic effects in various administration regimens. Such amounts are easily determined by those skilled in the art. The amount of protein will depend on the type and severity of the infection, the route of administration, etc. By "stabilizing buffer" is meant any of different buffer solutions which, when combined with the protein of the stabilized composition of the present invention, provide a stabilized protein composition, the composition of which allows therapeutic levels of such protein to be maintained by a prolonged period. The maintenance of therapeutic levels can be determined for example, by measuring protein activity, as determined by methods known in the art, preferably, the stabilizing buffer operates at physiological pH. Regulatory solutions include, but are not limited to, organic regulatory solutions, such as those zwitterionic regulatory solutions, generally referred to as "good buffer solutions" that operate in the range of 6 to 8.5. Examples of such stabilizing buffer solutions include: HEPES (N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid), TES (N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid) and TRICINE (N-tris (hydroxymethyl) methylene glycine), cacodylic acid, Bis (2-hydroxyethyl) -imino-tris ( hydroxymethyl) methane (BISTRIS), piperazin-N, N'bis- (2-ethanesulfonic acid) (PIPES), imidazole and tris (hydroxymethyl) amnomethane (TRIS). Example of regulatory solution that can be used in the stabilized protein compositions of the present invention are shown in table 2.

TABLE 2 The pH of the stabilized protein composition of the present invention may be in the range of 4.0 to about 8.

As used herein, the term "physiological pH", unless otherwise indicated, refers to the pH range found in mammals, including humans, cows, pigs, horses, goats, sheep, dogs and cats. The physiological pH of mammals is generally in the range of 6.5 to about 8.0. The temperature of the stabilized composition of the protein of the present invention may be in the range of 20 ° C to about 50 ° C. As used herein, the term "physiological temperature", unless otherwise indicated, refers to the range in body temperatures encountered in mammals, including humans, cows, pigs, horses, goats, sheep, dogs and cats. The physiological temperature of mammals are generally in the range of 37 ° C to about 41 ° C. The physiological temperatures of some example mammals are as follows: human, 37 ° C; cows, 39 ° C; cats, 38 ° C; dogs, 39 ° C; goats, 39 ° C; horses, 37 ° C; and pigs, 37 ° C. Preferably, the stabilized protein compositions of the present invention contain, as the G-CSF protein, and preferably, bovine G-CSF, in a stabilizing buffer solution, whose regulatory solution is selected from HEPES regulatory solution, TES regulatory solution and TRICINE regulatory solution. The resulting stabilized protein composition allows to maintain the activity of bG-CSF at therapeutically effective levels for a prolonged period of at least three days, at the physiological pH of cows and at the physiological temperature of cows at about 40 ° C. A stabilized protein composition can be prepared by combining the protein and stabilizing buffer using known and generally available combination techniques. A particular method for preparing a stabilized protein composition includes using the protein in a purified form, prepared in accordance with protein purification techniques known to those skilled in the art. For a particular protein of therapeutic value, the particular protein (up to its maximum solubility) can be dissolved in each of several pH regulators, such as HEPES, TES, TRICINE or other pH regulators, at varying concentrations of pH regulator, such as 0.05 to 2M. In addition, the pH of the solution can be varied, typically from about 4.0 to about 8.0. The maximum solubility of the protein in a particular pH regulator can be determined by conventional means known in the art. The solution can then be stored at the physiological temperature of a mammal in which the protein solution is to be administered, and the amount of protein present in the solution can be determined as a function of time. The therapeutic level of protein in the solution can be determined by monitoring the percent recovery of the protein as a function of time. The amount of protein remaining or percentage of protein recovery can be compared to a known (minimum) threshold value of protein that is required for therapeutic benefit. The sustained period can then be determined as the number of days during which the amount of protein remaining in the solution is equal to or greater than the known threshold value thereof that is required for therapeutic benefit. A pH regulator that is effective as a stabilizing pH regulator is one that, when combined with the particular protein, will provide sustained therapeutic levels of the protein over a sustained period, i.e., a longer period than is possible by administering the same protein to the mammal in the absence of the stabilizing pH regulator. The stability of a protein can be determined by measuring the activity of the protein as a function of time. The cleavage temperature (Tm) of the protein can be used as a marker of stability in solution and stability in vivo for the proteins. The splitting temperature of a particular protein refers to the temperature at which the protein loses its secondary structure and typically its activity, and can be determined using methods known to those skilled in the art, such as differential scanning calorimetry. The amounts of protein present in the stabilized protein compositions of the present invention may vary from about 0.1 mg / ml to about 5 mg / ml. For G-GSF, the preferred scale is from about 0.1 mg / ml to about 3 mg / ml. An example of a stabilized protein composition according to the present invention is a composition containing bG-CSF and pH regulator HEPES, wherein the bG-CSF is present at a concentration scale of about 0.1 to about 5 mg / ml, and where 'the pH regulator HEPES is present at a concentration scale of about 0.1 M to about 2M. More preferably, the concentration of bG-CSF is within the range of about 0.1 mg / ml to about 3 mg / ml. Another example of a stabilized protein composition according to the present invention, is a composition containing bG-CSF and pH regulator TES, wherein the bG-CSF is present at a concentration scale of about 0.1 to about 5 mg / ml, and where the pH regulator HEPES is present at a concentration scale of about 0.1 M to about 2M. More preferably, the concentration of bG-CSF is within the range of about 0.1 mg / ml to about 3 mg / ml. Yet another example of a stabilized protein composition according to the present invention, is a composition containing bG-CSF and TRICINE pH regulator, wherein the bG-CSF is present at a concentration scale of about 0.1 to about 5. mg / ml, and wherein the pH regulator HEPES is present at a concentration scale of about 0.1 M to about 2M. More preferably, the concentration of bG-CSF is within the range of about 0.1 mg / ml to about 3 mg / ml.

The stabilized protein composition of the present invention can be prepared in a frozen form or a lyophilized form using conventional means known to those skilled in the art. The lyophilized forms of the protein can be reconstituted with the stabilizing pH regulator. Alternatively, the solution can be stored in liquid form for immediate use. Preferably, the stabilized protein composition of the present invention is in a liquid form that maintains its long-term storage activity. The stabilized protein compositions of the present invention can be administered orally, parenterally (subcutaneously, intravascularly, intraperitoneally and intramuscularly), nasally, such as by inhalation, intraocularly or intradermally, or by infusion methods using forms known to those skilled in the art. The technique. Parenteral administration is preferred. Regardless of the route of administration, the stabilized protein compositions of the present invention can be formulated into pharmaceutically acceptable dosage forms by conventional methods known or that are apparent to those skilled in the art. The pharmaceutically acceptable dosage forms of the stabilized protein compositions of the present invention are preferably suitable for subcutaneous administration. A pharmaceutically acceptable dosage form for subcutaneous administration is typically of a volume no greater than about 20 ml (such as for administration to horses and livestock), is sterile (suitable for use in mammals) and, in addition, is well tolerated by the animal. mammal, that is, does not induce appreciable edema, pain or necrosis at the injection site. In general, the pharmaceutically acceptable dosage forms of the present invention may contain other pharmaceutically acceptable components such as, for example, surfactants or detergents, viscosity modifying agents, sugars or proteins, whose additional components are present in amounts suitable for pharmaceutical administration. effective and safe. For example, the pharmaceutically acceptable dosage form of the stabilized protein compositions of the present invention can be formulated following the accepted convention using suitable carriers, stabilizers, diluents and / or preservatives. The diluents may include water, saline, dextrose, ethanol, glycerol, and the like. Additives for isotonicity may include sodium chloride, dextrose, mannitol, sorbitol and lactose, among others. The stabilizers may include albumin, among others. Other suitable vehicles and additives are known to those skilled in the art, or will be apparent to them. The stabilized protein composition of the present invention can be provided in a kit, which comprises a first container having a therapeutically effective amount of a protein, and a second container having a pharmaceutically acceptable stabilizing pH regulator. The protein may be in solid form, such as a frozen or lyophilized form, or in liquid form. The stabilizing pH regulator can then be combined with the protein and administered to a mammal, such that the therapeutically effective amount of the protein from the first container, when combined with the pharmaceutically acceptable stabilizing pH regulator of the second container. , is able to maintain therapeutic levels of said protein in the mammal during a sustained period. Specific embodiments of the invention include a kit, wherein the protein is present in an amount sufficient to provide protection to a mammal for at least three days. The pharmaceutically acceptable dosage form of the present invention may be in the range of about 0.1 μg / kg to about 50 μg / kg, preferably from about 1 μg / kg to about 25 μg / kg, and more preferably around 3 μg / kg to approximately 25 μg / kg. The most preferred dosage form is about 24 μg / kg for use with bG-CSF. The dose is effective for at least about three days. The examples given below illustrate specific embodiments of the invention; however, the invention is not limited in scope to the examples given specifically.

EXAMPLE 1 Sustained stability of bG-CSF in pH regulators HEPES. TES AND TRICINE Concentrations of 0.1 M, 1 M and 2 M of pH regulator were prepared for each of three pH regulators, HEPES (N-2-hydroxyethylpiperazino-N-2-ethanesulfonic acid), TES (N-Tris (hydroxymethyl) acid). methyl-2-aminoethanesulfonic acid) and (TRICINE (N-Tris (hydroxymethyl) methylglycine) pH regulators were obtained from Fluka Biochemical USA The pH of each pH regulator was adjusted to 7.5 using sodium hydroxide (JT Baker, USA) The pH regulators were filtered sterile using a 0.2 micron GV filter (Millipore, USA) The concentrations of the pH regulators that were prepared were pH regulator HEPES: 0.1 M, 1 M and 2M pH regulator TES: 01 M, 1 M and 2M, and TRICINE pH regulator: 0.1 M, 1 M and 2 M. Solutions containing 0.1 mg / ml of bG-CSF were prepared in each of the pH regulators TES, TRICINE and HEPES in each of the pH regulator concentrations indicated above, adding an amount of 4.69 mg of global bG-CSF (based on a of 53.3%) was added to a 25 ml volumetric flask, which was then brought to volume with the appropriate concentration of pH regulator. Solutions were prepared containing 2 mg / ml of bG-CSF in each of the pH regulators TES, TRICINE and HEPES in each of the pH regulator concentrations indicated in Table 1, adding an amount of 93.8 mg of bG- Overall CSF (based on a 53.3% potency) to a 25 ml volumetric flask, which was then brought to volume with the appropriate concentration of pH regulator. The bG-CSF formulations were then filtered through a 0.22 micron low protein binding filter (Millipore G.V.). A volume of 1 ml of each formulation was placed in a 1 ml container, and then placed in an oven at 40 ° C for 9 days. The bG-CSF solutions stabilized with pH regulator that were prepared were 01 mg / ml of bG-CSF in (1) pH buffer HEPES: 0.1 M, 1 M and 2M; (2) pH regulator TES: 0.1 M, 1 M and 2M; and (3) TRICINE pH regulator: 0.1 M, 1 M and 2M. Samples were obtained from each of the containers every three days, and analyzed by means of size exclusion CLAR (CLAR-SEC). The results are shown in figures 1 to 6 and in tables 3 and 4.

TABLE 3 Table 3 shows the recovery percentage (remaining) of 01 mg / ml solutions of bG-CSF prepared as described above, as a function of time. The solutions were stored at 40 ° C.

TABLE 4 Table 4 shows the recovery percentage (remaining) of 2.0 mg / ml solutions of bG-CSF prepared as described above, as a function of time. The solutions were stored at 40 ° C. Figures 1 to 3 show the stability of 0.1 mg / ml solutions of bG-CSF at pH 7.5, under storage conditions of 40 ° C at varying concentrations of pH regulators HEPES, TES and TRICINE at 0.1 M, 1 M and 2M, respectively. The maintenance stability of the bG-CSF activity improved as the pH regulator concentration was increased to 1 M and more, as shown in Figures 1 to 3. At 0.1 mg / ml of bG-CSF, there was 90% of recovery of bG-CSF in HEPES at 1 M (Figure 1) - Figures 4 to 6 show the stability of 2.0 mg / ml solutions of bG-CSF at pH 7.5, under storage conditions of 40 ° C at varying concentrations of pH regulators HEPES, TES and TRICINE a 0. 1 M, and 1 M and 2M, respectively. Again, the stability or maintenance of the bG-CSF activity improved as the pH regulator concentration was increased to 1 M and more, as shown in Figures 4 to 6. The data presented in Tables 3 and 4 and Figures 1 to 6 show that the presence of the pH regulators HEPES, TES and TRICINE significantly maintain the activity of bG-CSF for sustained periods of 3 to 9 days.

EXAMPLE 2 Performance of bG-CSF in vivo formulated in water and pH regulators HEPES at 1 M, TES at 1 M and TRICINE at 1 M The live bG-CSF test formulated in water and pH regulators HEPES at 1 M, TES at 1 M and TRICINE at 1 M. was carried out in calves. A dose of 24 μg / kg was given to calves, and the numbers of PMN (neutrophils) were monitored. Figure 7 shows the PMN counts in peripheral blood (expressed as control percentage, value at 0 hours) for cattle treated with bG-CSF formulated in water, pH regulator HEPES at 1 M, 48 EUA) and pH regulator of bicarbonate. The results indicate that bG-CSF formulated in pH buffer HEPES at 1 M was the most stable of all the formulations tested, and exhibited stability similar to the pH regulator Neupogen® at pH 4.0. The stability of bG-CSF in pH regulator HEPES, as demonstrated in Figure 8, was surprising and unexpected, since it was previously known that bG-CSF was unstable at neutral or physiological pH conditions and at temperatures of about 40 ° C. or more. The bG-CSF in PBS at pH 7.0, Hanks pH regulator and bicarbonate pH regulator, was not stable. This was confirmed as described in table 5. TABLE 5 * Higher values correspond to lower activity. During 7 days of storage at 40 ° C, bG-CSF in Neupogen® and pH regulator HEPES did not lose any activity. As shown, bG-CSF in PBS was 10 times less active, and bG-CSF in Hanks pH buffer and bicarbonate was 100 to 1000 times less active than at the initial concentrations, respectively. 49 Figure 9 shows the effect of the concentration of the pH regulator HEPES on the stability of bG-CSF in pH regulator HEPES a 1000 mM, 500 mM, 100 mM, 50 mM, and 20 mM at 40 ° C. As shown in Figure 9, as the concentration of HEPES decreased, there was a significant loss in the stability of bG-CSF. Figure 10 is a thermogram of two different bG-CSF solutions. The upper thermogram, with a maximum temperature of 47 ° C, is for bG-CSF formulated in PBS at pH 7.5, and the lower thermogram, with a maximum temperature of 59 ° C, is for bG-CSF formulated in HEPES at 1 M at pH 7.5. In the absence of pH regulator HEPES, the splitting temperature of bG-CSF at pH 7.5 is approximately 40 ° C (start temperature), while bG-CSF in pH regulator HEPES a 1 M results in an increase of 10 ° C in the splitting temperature. An increase in the splitting temperature is indicative of stabilization.

EXAMPLE 4 Performance of bG-CSF in vivo formulated in HEPES at 1 M The in vivo bG-CSF test formulated in HEPES at 1 M was carried out in calves. A dose of 12 μg / kg was administered to calves, and the numbers of white blood cells (WBC) and PMN (neutrophils) were monitored. . The results are shown in figure 11. 50 Figure 1 1, which is a graph of the percentage of PMN (neutrophils) against time, is a comparison of three formulations: bG-CSF in water (as control), bG-CSF in HEPES at 1 M and bG-CSF in HEPES at 1 M plus poloxamer at 10%. As shown in Figure 11, PMN figures remain above the threshold (level associated with protection) for 3 days or 72 hours. Six calves were tested per formulation. In a second study, in which a dose of 24 μg / kg was administered to calves, using bG-CSF in pH buffer at 1 M plus 10% poloxamer, a single injection provided approximately 200 hours of protection, or approximately 8 days of coverage. This result shows that the pH regulator HEPES improves the stability of bG-CSF in vivo which, in turn, provides a period of sustained activity and, therefore, the supply of this protein.

EXAMPLE 5 Solubility of bG-CSF in HEPES at 1 M The solubility of bG-CSF in 1 M HEPES at pH 7.5 was determined. Approximately 80 mg of bG-CSF was dissolved in 30 ml of 1 M HEPES pH buffer (pH 7.5). The protein solution was filtered through a 0.2 micron Millipore GV filter, and then transferred to a 50 ml ultrafiltration cell. The cell was equipped with a low protein binding membrane with a molecular weight (MW) separation of 10., 000 The protein solution was concentrated using the ultrafiltration cell. At various time points, samples were taken from the cell for analysis by UV-Vis analysis at 310 nm (measured light scattering) and for concentration by CLAR RP. The absorbance at 310 nm was plotted against the concentration. The absorbance at 310 nm should increase linearly with the concentration; at saturation, there is a sudden interruption in the curve of 310 nm, and the absorbance at 310 nm increases dramatically. The concentration at which this occurs is the maximum solubility. This method is known to those skilled in the art, and is typically used to determine protein solubility. As shown in Figure 12, the maximum solubility of bG-CSF in 1 M HEPES at pH 7.5 is about 5 mg / ml. The maximum solubility of the protein is shown in the concentration that shows an interruption in the curve. At a concentration of approximately 5 mg / ml, there is a sudden increase in absorbance at 310 nm, which corresponds to the maximum solubility of the protein.

EXAMPLE 6 Effects of pH regulators HEPES. TES v TRICINE on the splitting temperature of bG-CSF Solutions were prepared containing 0.5 mg / ml bG-CSF in HEPES at 1 M, HEPES at 2M, TES at 1 M, TES at 2M and TRICINE at 1 M; and 2 mg / ml of bG-CSF in 1 M HEPES, 2 M HEPES, 1 M TES, 2 M TES and 52 to 1 M TRICINE. These solutions were prepared in the same manner as described in Example 1. A control solution was prepared using PBS (saline regulated at its pH with Dulbecco's phosphate, pH 7.4). The pH of the bG-CSF solutions was 7.5. The cleavage temperature of bG-CSF was determined by differential scanning calorimetry (Microcal Inc., E.U.A.) using a scanning rate of 60 degrees per hour at a temperature range of 20 ° C to 90 ° C. The results are shown in table 6.

TABLE 6 Temperature of splitting (0 ° C) The splitting temperature was used as a marker of the stability of the solution and the stability of the proteins in vivo. The results in Table 6 indicate that the splitting temperature (Tm) of bG-CSF formulated in pH regulators HEPES, TES or TRICINE at concentrations of 1 M and more was significantly higher compared to a PBS control. The three pH regulators raised the Tm by approximately 2 to 1 1 ° C. The concentration of pH regulator substantially affected the degree of increase in Tm. The Tm of bG-53 CSF increased as the pH regulator concentration also increased. There was an increase of about 3 ° C when the HÉPES concentration was increased from 1 M to 2M, and there was an increase of about 5 ° C when the TES concentration increased from 1 M to 2M. As the concentration of bG-CSF was increased, the Tm of bG-CSF decreased. There was a decrease of approximately 2 ° C when the concentration of bG-CSF was increased from 0.5 mg / ml to 2 mg / ml in pH regulators HEPES and TES. The pH regulator solution TES increased the Tm of bG-CSF by more than 1 1 ° C at a pH regulator concentration of 2M and a concentration of bG-CSF of 0.5 mg / ml. The results of table 6 show that the three pH regulators (HEPES, TES and TRICINE) significantly increase the Tm of bG-CSF compared to PBS. bG-CSF formulated in 2M TES exhibits the highest stability in solution with respect to the other pH regulators.

EXAMPLE 7 Comparison of the stability of human and bovine G-CSF in formulations of PBS v HEPES Formulations of 0.15 mg / ml of hG-CSF and bG-CSF were prepared in pH regulated saline with phosphate (Dulbecco's PBS, pH 7.4), and pH regulator HEPES at 1 M (1 M HEPES, pH 7.5 ). The formulations were placed in 1 ml containers (filling volume of 400 54 ml), and stored at 40 ° C for 10 days. Samples were tested every three days by size exclusion chromatography (CLAR SEC), and they were visually inspected. Figure 13 shows that a significant improvement in the stability of human G-CSF was observed when formulated in pH regulator HEPES at 1 M, when compared with PBS. Human G-CSF exhibited degradation for more than 10 days at 40 ° C when formulated in PBS at pH 7.4, while a recovery of 65% was observed when formulated in pH buffer HEPES at 1 M. Figures 13 and 14 show that bovine G-CSF exhibits slightly better stability in HEPES and PBS formulations than human G-CSF. A recovery of approximately 80% bovine G-CSF in the 1 M HEPES formulation was observed after 10 days at 40 ° C, while a recovery of approximately 65% of human G-CSF was observed. Both proteins, bovine and human G-CSF, are substantially more stable in pH buffer HEPES at 1 M, than in PBS. 55 EXAMPLE 8 Stability of human and bovine G-CSF in formulations of 1 M HEPES Formulations of 0.1 mg / ml of hG-CSF and bG-CSF were prepared in 1 M HEPES pH buffer (1 M HEPES, pH 7.5). The formulations were placed in 1 ml containers (400 ml filling volume), and stored at 40 ° C for 10 days. Samples were tested every three days by size exclusion chromatography (CLAR SEC), and inspected visually. Figure 15 shows the stability of bovine G-CSF and human G-CSF in formulations of HEPES at 1 M. There was a recovery of approximately 90% of bovine G-CSF, and a recovery of approximately 70% of G-CSF. Human CSF after 10 days at 40 ° C.

EXAMPLE 9 Effect of the pH of the formulation on the stability of bG-CSF Formulations of 0.1 mg / ml solutions of bG-CSF were prepared in pH regulators HEPES at 1 M and TES at 1 M at pH 4.0 and 7.5. The formulations were placed in 1 ml containers (400 ml filling volume), and stored at 40 ° C for 10 days. The samples were put to 56 test every three days by size exclusion chromatography (CLAR SEC) As shown in Figures 16 and 18, recovery of bG-CSF after 10 days at 40 ° C was approximately 100% at pH 4.0, comparing with Figures 17 and 19, which show a recovery of approximately 80 to 85 % observed when bG-CSF was formulated at pH 7.5.

EXAMPLE 10 Six-month sampling of the long-term thermal stability study of bG-CSF in 1.0 M HEPES and TES formulations The formulations included in this study are given below. A commercial formulation of 1.0 M HEPES was obtained from GibcoBRL (Lot # 1016436), while 1.0 M TES was prepared from powder obtained from Fluka Scientific (Lot # RA12602). The pH values of the pH regulator were adjusted to 7.5. BG-CSF was provided by Bioprocess (Lot # BP185-1 1) at a purity of 53.3%. Formulations of 0.1 mg / ml and 2.0 mg / ml of bG-CSF were prepared in pH buffer HEPES at 1.0 M and TES at 1.0 M. For the storage of samples, containers were used.

Flint type 1 3.5 ml (Lot # R04105-7322) with 1888 Gray T / F 13 mm plugs (Lot # R05619-7487) using a 1.0 ml fill volume. A summary of sample extraction and storage points is given in Table 7. Five containers of each formulation were stored for each test time point.

TABLE 7 Summary of time points for power analysis and storage of samples Three samples were obtained from each formulation of each storage chamber (5, 30 and 40 ° C), and were tested by CLAR RP and SE for bG-CSF potency. Each sample was tested three times. The concentrations were calculated using a standard curve which was previously determined. The percentage (%) of the initial concentrations of bG-CSF for each of the analyzes was determined, and a mean (average) was calculated for each formulation at each time point. The average% of the initial concentrations of bG-CSF was plotted against the time for each storage temperature to graphically show the decrease in bG-CSF power. Figures 20 and 21 are the results obtained by CLAR RP and SE, respectively, for samples stored at 5 ° C. The results obtained for samples stored at 30 ° C are given in Figures 22 and 23, while those obtained 58 for samples stored at 40 ° C can be found in Figures 24 and 25. There was little degradation of bG-CSF in samples stored at and 30 ° C, excluding the formulation of 0.1 mg / ml of protein in pH regulator TES at l .O M.

EXAMPLE 11 Stabilization capacities of the pH regulator HEPES over biotherapeutic proteins The TM values of the proteins of interest described below were determined in phosphate pH regulators and HEPES using a MicroCal Model VP-DSC microcalorimeter. A phosphate pH regulator solution at 25 mM was prepared using Na2HP04 (batch number 08019PQ) from Aldrich, and the pH was adjusted to 7.5. The pH regulator HEPES at 1.0 M (pH = 7.5, lot number 1016436) was obtained from GibcoBRL. PH regulator exchanges were carried out by a stirred ultrafiltration cell model 8010 (Amicon, Inc.), in combination with a YM10 or YM10 ultrafiltration membrane Diaflo® (Amicon, Inc.), depending on the size of the protein. 2.0 mg of lyophilized pST (batch number 41509-217-2) obtained from Bioprocess in 2.0 ml of Milli-Q water were reconstituted. After five exchanges, 1 ml of the reconstituted protein was transferred into phosphate pH regulator 59 at 25 mM using a YM10 membrane. The remaining ml was then exchanged in pH regulator HEPES at 1.0 M. Samples were prepared at a concentration of 1.0 mg / ml protein, and were then analyzed by microcalorimetry. The analysis of PST by microcalorimetry was carried out twice in phosphate pH regulators and HEPES. Approximately 2.0 ml of NIF (lot number 440631-22-7) was obtained from Bioprocess at a concentration of 2.97. The sample received from Bioprocess was divided into two aliquots. 1.0 ml of the protein was exchanged in pH buffer of phosphate at 25 mM, and the remaining NIF was exchanged in pH buffer HEPES at 1.0 M. In each case, 5 exchanges were used using a YM30 membrane. Solutions at a concentration of 1.0 mg / ml were prepared in the appropriate pH regulator, and the Tm values were determined by microcalorimetry. The biotherapeutic proteins that were studied are included in Table 8 with their respective Tm values that were determined for phosphate pH regulators and HEPES.

TABLE 8 Results of Tm obtained in the microcalorimetry study of four biotherapeutic proteins in phosphate pH regulators and HEPES 60 Table 8 shows that the pH regulator HEPES provides increased stability for NIF and pST.

EXAMPLE 12 Sustained live activity of recombinant bovine granulocyte colony stimulating factor (rbG CSF) using pH regulator HEPES Bovine granulocyte colony stimulating factor (bG-CSF) was obtained from Bioprocess Research and Development-Pfízer (Groton, CT), mannitol from EM Industries (Hawthorne, NY), phosphate buffered saline (PBS) Dulbecco 1X from GibcoBRL (Grand Island, NY), sodium citrate and sodium acetate from Aldrich (Milwaukee, Wl), Tween-80, sodium chloride and hydrochloric acid from JT Baker (Phillipsburg, USA).

CLAR RP and size exclusion chromatography (SEC) The stability of the solution was monitored by CLAR RP and SEC. CLAR RP was carried out using a C4 column for Vydac protein, using a mobile phase of 0.1% H20-FA (solvent A) and 0.1% CAN-TFA (solvent B). Flow rate: 1 ml / min; UV detection: 220 nm; Temperature: 25 ° C. Size exclusion chromatography was carried out using a TosoHaas, TSK-GEL SW? _, 7.8 mm ID x 30 cm column. Phase 61 mobile: NaCl at 0.3 M in 0.05 M citrate pH regulator, pH 5.75; flow rate: 1 ml / min; UV detection: 280 nm; Temperature: 25 ° C.

Microcalorimetry The denaturing temperature (TD) was measured using a DSC VP system (MicroCal, Inc.). About 1 ml of the solution was loaded into the cell, and run against a reference placebo formulation at a rate of about 10 ° C / min.

Circular dichroism Secondary structure of bG-CSF was monitored using circular dichroism spectroscopy equipment with a scanning temperature measurement accessory (CD * ORD Model J-710/720-Japan Spectroscopic Co., LTD).

Bioensavo The in vitro activity of bG-CSF formulations was determined using a murine bone marrow cell proliferation test (BMC test). Bone marrow cells were harvested aseptically from the femurs of female CF1 mice (Charles River) by removing the femur and gently squirting the marrow out of the bone using a 3cc 23G syringe and Hanks balanced salt solution (Gibco BRL). The cell suspension was filtered through a nylon screen to remove 62 residues, and then centrifuged at 1100 rpm for 10 minutes at room temperature. The supernatant was discarded, and the pellet was resuspended in 15 ml of RPM1 medium (Gibco BRL) supplemented with 10% fetal bovine serum.

(Gibco BRL), 1% penicillin-streptomycin (10,000 units / ml) and 1% L-glutamine (Gilbco BRQ). The number of cells in the bone marrow was quantified using a 256 Coulter channeler, and the cell concentration was adjusted to yield 6.67 x 10 5 cells / ml. About 105 cells were added to each well of a 96-well plate. Bovine granulocyte colony stimulating factor formulations were then added to each well (in triplicate) at various concentrations. After an incubation period of 3 days at 37 ° C (CO2 at 5%), 3H-thymidine (New England Nuclear, Boston, Mass) was added to each well at a final concentration of 2μCi / ml. The radiometric labeling was allowed to proceed for at least 18 hours at 37 ° C (C02 at 5%). Plates were frozen at -20 ° C, thawed, and cells harvested on 96-well glass fiber mats using a Brandel cell harvester (Biomedical Research and Development Laboratories, Gaithersburg, Maryland). Activity was determined using a 1205 Betaplate liquid scintillation counter (Wallac, Gaithersburg, Maryland). The activity of the bG-CSF formulations was determined by dividing the sample counts per minute by means of the control counts of the media per minute (number of times on the above). The activity of 3 or more times above was considered positive. 63 In-vivo bG-CSF activity The live activity of bG-CSF formulations in young bred calves varying from approximately 100 to 150 kg body weight was tested. The calves were purchased and shipped to the Animal Health Research Center in Terre Haute, Indiana, and acclimated to the facility for a minimum period of two days before being assigned to a study. Most calves were used for a study, allowed to rest for at least a week, and then reassigned to a second study. The calves were not used for more than two studies. Before being assigned to a study, the calves were pre-examined 1 to 3 days before starting the study, evaluating their rectal temperature, coforal weight, general health and differentials and total white blood cell counts (WBC). In general, calves with rectal temperatures > were excluded from the studies; 104 ° C and WBC total counts of > 4000 / mm3 or 12,000 / mm3. On day 0, calves were bled and weighed before treatment. The dose of 24 μg / kg of the formulation of bG-CSF at the time of treatment for each calf was calculated based on body weight and administered by subcutaneous injection in the pre-scapular region of the neck. Blood samples were collected in EDTA anticoagulant for WBC differentials by venipuncture of the neck at predetermined times after treatment. Total WBC counts were carried out in a cell counter for Nova Celltrak I hematology using a 1: 250 dilution of whole blood in isotonic diluent. The 64 differentials of the WBC counts were carried out using dehydrated blood smears stained with a Diff-Quik (Dade) staining kit. The count of a total of 100 white blood cells was counted, and they were differentiated in a Zeiss optical microscope with a 100x oil immersion lens and 12.5x eyepieces (total magnification = 1250x).

Effect of pH and temperature on the stability of bG-CSF in solution Table 9 shows the effect of temperature on the stability of bG-CSF. The impact of temperature on the stability of bG-CSF in solution was followed by CLAR RP, CLAR SEC, bioassay and visual inspection (formulation: 0.1 mg / ml bG-CSF, mannitol a %, pH regulator of acetate at 10 mM, Tween-80 at 0.004%, pH 4.0 As observed in table 9, there is a discontinuity in the stability of bG-CSF at temperatures of 40 ° C or more. By CLAR RP and CLAR SEC there is a loss of the apparent maximum protein value at 40 ° C and above. The disappearance of the apparent maximum value at higher temperatures is followed by an increase in the particles in the solution. This was observed visually and monitoring the scattering of light at 310 nm. The bovine G-CSF solutions stored at 40 ° C for 3 weeks are 10 to 100 times less active than at 5 ° C and 30 ° C. At 50 ° C for 3 weeks, bG-CSF is 100 to 1000 times less active than solutions stored at 5 ° C and ° C. 65 TABLE 9 Stability of bG-CSF as a function of temperature (stability for 3 weeks) * Activity tested in BMC test in mice (note: higher figures indicate less activity). Circular dichroism (CD) was used to track the impact of temperature on bG-CSF. The CD spectrum of bG-CSF is illustrated in Figure 26. The spectrum suggests that the secondary structure of bG-CSF is primarily an alpha-helix, similar to human G-CSF, which is structurally very similar to bG- CSF The CD spectra were examined at various temperatures to determine the denaturing temperature (TD). Figure 27 is a graph of the molar ellipticity at a wavelength of 222 nm (characteristic wavelength for an alpha-helix) as a function of temperature. Between 40 ° C and 50 ° C, the molar ellipticity increases, indicating the loss of secondary structure and the denaturation of bG-CSF. 66 The effect of pH on the stability of bG-CSF in solution was also evaluated. To delineate only the effect of pH on the stability and not the impact of denaturation due to temperature, the solutions were stored at 30 ° C (TD of bG-CSF between 40 ° C and 50 ° C). Table 10 summarizes the stability of bG-CSF as a function of pH over a period of 2 weeks at 30 ° C. The rate of loss of protein activity increases as the pH increases. The data suggest that at low pH, the cysteine in bG-CSF is protonated and, consequently, the formulation is more stable. At high pH, this free cysteine intervenes in the disulfide exchange reactions and is the probable cause of instability.

TABLE 10 Stability of bG-CSF as a function of pH (stability for 2 weeks at 30 ° C) * Activity tested in BMC test in mice (note: higher figures indicate less activity). 67 Effect of the pH regulator HEPES on the stability of bG-CSF in solution It has been observed that bG-CSF formulated in pH buffer HEPES at 1 M at pH 7.5 exhibits greater stability in solution even when stored at 40 ° C during several days. This was unexpected, since it was previously known that bG-CSF is unstable at neutral pH and denatures at temperatures of 40 ° C or more. Figure 28 illustrates the effect of the HEPES pH regulator concentration on the stability of bG-CSF in solution at pH 7.5 and storage temperature of 40 ° C. The stability of bG-CSF decreased significantly as the concentration of pH regulator HEPES decreased. The effect of 1 M HEPES on the denaturation temperature (TD) of bG-CSF was determined by microcalorimetry. Figure 10, a thermogram, compares the two formulations (with and without HEPES at 1 M) on the TD of bG-CSF. In the absence of pH regulator HEPES, the start of the endothermic transition is about 40 ° C, while bG-CSF formulated in 1 M HEPES pH regulator has a TD start of around 50 ° C. An increase in denaturation temperature is usually indicative of stabilization.

Activity of bG-CSF in vivo The activity of this formulation was evaluated in vivo in cows. Figure 29, which is a graph of the WBC count against time, is a comparison of bG-CSF formulated in HEPES at 1 M, comparatively with 68 the "control" formulation. "Control" refers to the formulation containing 5% mannitol, pH buffer of acetate at 10 mM and Tween 80 at pH 4.0. As seen in Figure 29, the WBC count remains above the threshold value of the 200% baseline level (level associated with infection protection) for only about 24 to 30 hours.

However, when bG-CSF was formulated in 1 M HEPES, the PMN figures remained above the threshold value for a minimum of 3 days or 72 hours (In some cases, the WTC remained above the threshold value for almost a week). This study could be reproduced (in each study, 6 cows were used per formulation). The results of this study suggest that the HEPES pH regulator not only functions as an in vitro stabilizer, but in some way improves the performance of bG-CSF in vivo. The unexpected results observed with the pH regulator HEPES on the performance of bG-CSF have led to the investigation of similar pH regulators, such as MOPS, HEPPS, TES and TRICINE. These pH regulators also exhibited a stability of bG-CSF improved in vitro similar to that of the pH regulator HEPES. An in vivo study was carried out, where bG-CSF was formulated in pH regulator TES and TRICINE pH regulator; both formulations resulted in a prolonged activity of bG-CSF in vivo in cows, similar to the HEPES formulation. The formulation of bG-CSF in 1 M HEPES pH regulator produces sustained bG-CSF activity in vivo. This sustained activity 69 may be a result of improved stability of bG-CSF at the injection site. The stability of bG-CSF in solution at neutral pH and temperature of 40 ° C improved significantly when bG-CSF was formulated in HEPES at 1 M. Other organic pH regulators, such as MOPS, HEPPS, TES and TRICINE, also gave an improvement in the stability of bG-CSF.

Claims (26)

70 NOVELTY OF THE INVENTION CLAIMS

1. - A stabilized protein composition comprising a potein and a stabilizing pH regulator, whose composition is capable of maintaining therapeutic levels of said protein during a sustained period.

2. The composition according to claim 1, further characterized in that the protein is selected from the group consisting of: stimulation factors of colonies, somatotropins, cytokines, antibodies and antigens.

3. The composition according to claim 2, further characterized in that the protein is a colony stimulation factor.

4. The composition according to claim 3, further characterized in that the protein is selected from the group consisting of: human G-CSF, bovine G-CSF and canine G-CSF.

5. The composition according to claim 4, further characterized in that the protein is bovine G-CSF.

6. The composition according to claims 1 or 5, further characterized in that the composition is at a physiological pH.

7. The composition according to claim 1 or 5, further characterized in that the composition is at a pH of about

4. 0 to approximately 7.5. 8. The composition according to claims 1 or 5, further characterized in that the composition is at a physiological temperature.

9. The composition according to claim 1 or 5, further characterized in that the stabilizing pH regulator is selected from the group consisting of: HEPES, TES and TRICINE.

10. The composition according to claim 1 or 5, further characterized because the sustained period is at least about 3 days.

11. The composition according to claim 1 or 5, further characterized because the sustained period is at least about 3 days in vivo.

12. The composition according to claim 4 or 5, further characterized in that G-CSF is present at a concentration in the range of 0.01 to 5 mg / ml.

13. The composition according to claims 1, 4 or 5, further characterized in that the stabilizing pH regulator is present at a concentration ranging from about 0.05 M to about 2 M.

14. The composition according to claim 5, further characterized in that the stabilizing pH regulator is HEPES, and is present at a concentration of about 1 M.

15. A pharmaceutically acceptable dosage form of a protein composition. stabilized for parenteral administration to a mammal, characterized in that it comprises a protein and a pharmaceutically acceptable stabilizing pH regulator, whose composition is capable of maintaining therapeutic levels of said protein during a sustained period, wherein the protein is present in an amount sufficient to provide a therapeutic benefit to a mammal during a predetermined period.

16. The pharmaceutically acceptable dosage form according to claim 15, further characterized in that it comprises a component selected from the group consisting of viscosity modifiers and surfactants.

17. The pharmaceutically acceptable dosage form according to claim 15, further characterized in that the protein is bovine G-CSF present at a concentration in the range of about 0.01 to 5 mg / ml, the stabilizing pH regulator is selects from the group consisting of HEPES, TES and TRICINE, the mammal is a cow, the predetermined period is at least about 3 days, and the composition is at a pH of about 7.5.

18. The pharmaceutically acceptable dosage form according to claim 17, further characterized in that the pH regulator is HEPES, wherein the HEPES is present at a concentration ranging from about 0.05 M to about 2 M.

19. - The pharmaceutically acceptable dosage form according to claim 18, further characterized in that the bovine G-CSF is administered at a dose in the range of about 0.1 μg / kg to about 50 μg / kg.

20. A method for preparing a pharmaceutically acceptable dosage form of a stabilized protein composition for parenteral administration to a mammal, characterized in that it comprises the step of combining a protein and a stabilizing pH regulator, whose stabilized protein composition is capable of of maintaining therapeutic levels of said protein during a sustained period, wherein the protein is present in an amount sufficient to provide protection to a mammal for at least about 3 days.

21. A method for treating or preventing infections in mammals, characterized in that it comprises administering to the mammal a therapeutically effective amount of a stabilized protein composition, wherein the stabilized protein composition comprises a protein and a stabilizing pH regulator, whose composition is able to maintain therapeutic levels of said protein for a sustained period of at least about 3 days.

22. A method for treating or preventing mastitis, metritis or bovine respiratory disease in cattle, characterized in that it comprises administering to the cow a therapeutically effective amount of a stabilized G-CSF composition, wherein the composition of stabilized G-CSF it comprises G-CSF and a stabilizing pH regulator, whose composition is capable of maintaining therapeutic levels of said protein for a sustained period of at least about 3 days.

23. A method for maintaining therapeutic levels of a protein in a mammal during a sustained period, characterized in that it comprises administering to the mammal a stabilized protein composition, wherein the stabilized protein composition comprises a protein and a stabilizing pH regulator, whose composition is capable of maintaining therapeutic levels of said protein for a sustained period of at least about 3 days.

24. The method according to claims 20, 21, 22 or 23, further characterized in that the protein is bovine G-CSF present at a concentration in the range of about 0.01 to 5 mg / ml, the stabilization buffer is selected from the group consisting of HEPES, TES and TRICINE, and the composition is at a pH of about 7.5.

25. A kit for administering to the mammal a stabilized protein composition, comprising a first container having a therapeutically effective amount of a protein, and a second container 75 having a pharmaceutically acceptable stabilizing pH regulator, characterized in that the amount Therapeutically effective protein of the first container, when combined with the pharmaceutically acceptable stabilizing pH regulator of the second container, is capable of maintaining therapeutic levels of said protein in the mammal for a sustained period of at least about 3 days.

26. The equipment according to claim 25, further characterized in that the protein is bovine G-CSF present at a concentration in the range of approximately 0.01 to 5 mg / ml, the stabilizing pH regulator is selected from the group that consists of HEPES, TES and TRICINE, and the composition is at a pH of about 7.5. 27.- A stabilized protein composition comprising bovine G-CSF and pH regulator HEPES, whose composition is capable of providing a prolonged storage life in the scale of around 3 weeks to approximately 18 months. 28. The composition according to claim 27, further characterized in that the pH regulator HEPES is at a concentration ranging from about 0.05 M to about 2 M, the composition is at a pH of about 7.5, and the temperature of the composition is less than about 40 ° C. 29. The composition according to claim 28, further characterized in that the temperature is about 4 ° C.