HK1026151B - Stabilized protein compositions - Google Patents

Description

Stabilized protein compositions

The present invention relates to stabilized protein compositions. The stabilized compositions of the invention can be used to deliver therapeutically effective amounts of proteins including colony stimulating factors, such as bovine granulocyte colony-stimulating factor (bG-CSF), to mammals, including humans, cows, pigs, horses, goats, sheep, dogs and cats, over an extended period of time. More specifically, the stabilized compositions of the invention contain stabilizing buffers such as HEPES, TES and TRICINE, which are capable of maintaining protein activity for extended periods of time in vivo and in vitro.

The preparation of therapeutically effective protein preparations with in vivo and in vitro activity with extended shelf life, such as G-CSF, remains a problem. Such protein preparations must maintain their activity and biological integrity for a suitable period of time to ensure effective treatment. In addition, such protein preparations must be preparable and capable of being administered to an animal in a pharmaceutically acceptable manner.

Pharmaceutical compositions of proteins have been provided in frozen or lyophilized form for in vitro storage under storage conditions that provide for extended periods of protein activity. The lyophilized preparation is reconstituted with a pharmaceutically acceptable diluent (e.g., sterile water for injection) prior to use. Pharmaceutical compositions of proteins are also provided in liquid form. Such liquid protein formulations are difficult to store, particularly at high temperatures, due to loss of activity of the protein over time.

Therapeutically effective protein formulations, whether in solid (lyophilized) or liquid form, are difficult to administer to an animal in a manner that does not result in sudden inactivation after administration, such as by subcutaneous injection. The rapid inactivation of proteins at the injection site makes them inconvenient for use in the treatment of mammalian infections, since effective treatment requires a daily dosage to be guaranteed during the required recovery period. Granulocyte colony stimulating factors (G-CSFs), such as bovine granulocyte colony stimulating factor (bG-CSF), are unstable at 40 ℃ or higher due to loss of secondary structure, disulfide exchange and thus cause inactivation because the body temperature of cattle is about 40 ℃ and the injection site is in the physiological pH range, where inactivation occurs.

A variety of protein formulations having extended shelf life are known. U.S. Pat. No. 5,104,651(Boone et al, issued on 1992, month 4 and 14) relates to a pharmaceutical composition of G-CSF and an acid having a pH of 3.0-3.7, which has an electrical conductivity of less than 1000. mu. mhos/cm. U.S. Pat. No. 4,992,271(Fernandes et al, issued 2/12 1991) relates to a pharmaceutical composition comprising a biologically active recombinant interleukin-2 protein dissolved in an aqueous carrier medium at pH6.8-7.8 and a stabilizer for the protein (e.g., human serum albumin). U.S. Pat. No. 4,623,717(Fernandes et al, issued 11/18 1986) relates to pasteurised therapeutically active protein compositions which are pasteurised by mixing with the protein prior to sterilisation an amount of sugar or reducing sugar and an amino acid which produces a stabilising effect. U.S. Pat. No. 4,645,830(Yasushi et al, 24.2.1987) relates to a stable interleukin 2 composition comprising interleukin 2, human serum albumin and a reducing compound in a solution at a pH of 3 to 6. U.S. patent 4,647,454(Cymbalista, 3.3.1987) relates to a method for stabilizing human fibroblast interferon with polyvinylpyrrolidone. U.S. patent 4,675,184(Hasegawa et al, issued 6/23 1987) relates to a pharmaceutical composition for treating viral infections comprising an interferon, a tri-or polyhydric sugar alcohol, an organic buffer and a pharmaceutically acceptable carrier or diluent, wherein the composition has a pH of about 3 to 6. The above documents are incorporated by reference in their entirety.

One example of a therapeutically effective protein is granulocyte colony stimulating factor (G-CSFs). Granulocyte colony stimulating factor (G-CSF) is one of several glycoprotein growth factors, known as colony stimulating factors. Such colony stimulating factors support the proliferation of hematopoietic progenitor cells and stimulate the proliferation of specific bone marrow precursor cells and their differentiation to form granulocytes. In addition, G-CSF is able to stimulate the formation of neutrophil colonies and induce late differentiation of murine myelomonocytic leukemia cells in vitro. G-CSF has also been shown to stimulate neutrophil functional activity leading to enhanced microbicidal activity. G-CSF is known to have an amino acid sequence of 174 amino acids.

Recombinant forms of CSFs and G-CSFs have been prepared. The cloning and expression of DNA encoding human G-CSF is a known technique (Nagata, S et al, Nature 319: 415-418 (1986)). WO-A-8604606 and WO-A-8604506 describe genes encoding human G-CSF. U.S. patent 5,606,024(Boone et al, issued on 25/2/1997) and U.S. patent 5,472,857 (issued on 5/12/1995) describe DNA sequences encoding canine granulocyte colony-stimulating factor (cG-CSF) and a method of treating or inhibiting infection in canines or felines by administering to these animals an effective amount of human and canine G-CSF. U.S. Pat. No. 4,810,643(Souza, granted on 3/7 1989) describes human G-CSF-like polypeptides. European patent application 719860 (published 3.7.1996) describes the amino acid sequence of naturally occurring bovine granulocyte colony stimulating factor (bG-CSF), DNA sequences encoding bG-CSF, and methods of treating or inhibiting mastitis in an animal by administering to the animal an effective amount of G-CSF. WO-A-8702060 describes human G-CSF-like polypeptides, their coding sequences and methods of preparation. U.S. Pat. No. 4,833,127(Ono et al, 5/23 (1989)) describes a novel bioactive human granulocyte colony stimulating factor. European patent application 612846 (published on 8/31 of 1994) describes certain G-CSF analogues and compositions containing them (the entire contents of which are incorporated herein by reference).

Granulocyte colony stimulating factor can be used as an anti-infective agent that enhances the immunity of an animal, rather than acting on a specific microbial target essential for growth or toxicity. Few other commercial veterinary agents act on non-specific immune responses, thereby increasing resistance to microbial infection. The existing effective control methods are limited to conventional antibacterial drugs and a limited number of biologicals. Economic losses due to milk loss limit the use of conventional antibacterial drugs. Current vaccines only work on a limited number of species and the potency of these agents varies widely in the field. The most successful vaccines (E.coli J5) have limited their use worldwide due to safety issues caused by endotoxin contamination.

Mastitis is a major disease affecting dairy producers worldwide. In the united states, the annual economic loss due to mastitis exceeds 10 billion dollars. Losses relate to mortality, milk discard, rapid and slow reduction in milk production, increased labor costs for cattle killed early in the year, as well as veterinary and livestock husbandry. Perinatal cows have a reduced immunoreactivity (neutrophil function), which makes their mammary glands more susceptible to bacterial infection. The effect of increased susceptibility may be explained by the fact that approximately 40% of emerging clinical intramammary infections occur in the first two weeks after delivery. Mastitis is associated with a wide variety of pathogenic bacteria, including gram-positive and gram-negative bacteria. Pathogenic microorganisms known to cause mastitis are Escherichia coli (Escherichia coli), Staphylococcus aureus (Staphylococcus aureus), Streptococcus agalactiae (Streptococcus agalactiae), Streptococcus uberis (Streptococcus uberis), Streptococcus dysgalactiae (Streptococcus dysgalactiae), Aerobacter aerogenes (Aerobacter aeogens), Klebsiella pneumoniae (Klebsiella pneumoniae), and Pseudomonas aeruginosa (Pseudomonas aeruginosa). These pathogenic bacteria enter the udder through the milk duct, causing inflammation of the milk-producing tissue, resulting in the formation of scar tissue, resulting in permanent loss of milk production. Various forms of mastitis include: mastitis, chronic mastitis, clinical mastitis, and subclinical mastitis.

Current antibiotic therapies and vaccines have a number of drawbacks that limit their use in lactating cows. It has been found that mastitis control with antibiotic therapy is insufficient. There is therefore a need for a biotherapeutic agent that will help restore normal immunity and reduce the incidence and severity of mastitis.

Bovine respiratory disease, also known as typhus, is another common disease that infects cattle. Cattle are exposed to respiratory diseases after shipping to a feedlot or pasture due to various stresses including weaning, castration, cutting corners, ban, crowding, exposure to infectious substances, diet and temperature changes, as well as infection by certain known pathogenic bacteria. Pasteurella haemolytica (Pasteurella haemolytica) is a common pathogenic bacterium that causes damage to the bovine respiratory system.

Several other infectious diseases, including various reproductive diseases, are also known to infect humans, pigs, cattle, dogs, cats, horses, goats, sheep. One example of such a disease (which occurs in cattle) is metritis.

There is therefore a need for a stable protein composition which maintains an extended period of therapy in vivo. In addition, there is a need for protein formulations with extended in vitro storage shelf life and shelf life.

The present invention relates to a stabilized protein composition comprising a protein and a stabilizing buffer, said composition being capable of maintaining a therapeutic level of the protein over a sustained period.

Particular embodiments of the present invention include a stabilized protein composition that is at physiological pH.

Other embodiments of the invention include a stabilized protein composition, which is at physiological temperature.

Other embodiments of the invention include a stabilized protein composition wherein the stabilizing buffer is selected from the group consisting of HEPES, TES and TRICINE.

Still other embodiments of the present invention include a stabilized protein composition wherein the sustained period is at least about 3 days.

Still other embodiments of the present invention include a stabilized protein composition, wherein the protein is selected from the group consisting of a colony stimulating factor, a growth hormone, an interleukin, an interferon, a cytokine, an antibody, and an antigen.

More specific embodiments of the present 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.

A more specific embodiment of the invention comprises a stabilized protein composition wherein the protein is G-CSF at a concentration in the range of 0.01 to 5 mg/ml.

Another embodiment of the invention includes a stabilized protein composition wherein the protein is G-CCF and the stabilizing buffer is selected from the group consisting of HEPES, TES and TRICINE.

Other more specific embodiments of the invention include a stabilized protein composition wherein the protein is G-CSF and the concentration of the stabilizing buffer is in the range of about 0.05M to about 2M.

Other embodiments of the invention include a stabilized protein composition wherein the protein is G-CSF and the composition is at physiological pH.

Other embodiments of the invention include a stabilized protein composition, wherein the protein is G-CSF and the composition is at physiological temperature.

A more specific embodiment of the invention encompasses a stabilized protein composition wherein the protein is bovine G-CSF.

Other embodiments of the invention include a stabilized protein composition wherein the protein is bovine G-CSF at a concentration in the range of 0.01 to 5mg/ml bG-CSF.

Other embodiments of the invention include a stabilized protein composition wherein the protein is bovine G-CSF and the stabilizing buffer is selected from the group consisting of HEPES, TES and TRICINE.

Other embodiments of the invention include a stabilized protein composition wherein the protein is bovine G-CSF and the concentration of the stabilizing buffer is in the range of about 0.0SM to about 2M.

Other embodiments of the invention include a stabilized protein composition wherein the protein is bovine G-CSF and the composition is at physiological pH.

Other embodiments of the invention include a stabilized protein composition wherein the protein is bovine G-CSF and the composition is at physiological temperature.

Preferably, the stabilized protein composition of the invention is a composition comprising bovine G-CSF dissolved in HEPES buffer. More preferably, the HEPES buffer concentration is from about 0.05M to about 2M. The bovine G-CSF formulation is preferably at physiological pH (e.g., 7.5). Moreover, such preferred bovine G-CSF formulations are capable of maintaining therapeutic levels of bovine G-CSF for extended periods of time (from about at least 3 days to 7 days or longer).

In addition, the stabilized protein composition of the present invention is a composition comprising bovine G-CSF dissolved in HEPES buffer, which composition ensures extended shelf-life and shelf-life. Preferably, the HEPES buffer concentration is from about 0.05M to about 2M. More preferably, the composition is maintained at a pH of from about 4.0 to about 7.5, preferably 4.0, and at a temperature of less than about 40℃, preferably about 4℃. The extended shelf life and shelf life is from about 3 weeks to about 18 months, preferably, from about 6 weeks to about 1 year.

The invention also relates to a pharmaceutically acceptable dosage form of a stabilized protein composition for parenteral administration to a mammal, comprising a protein and a pharmaceutically acceptable stabilizing buffer, which composition is capable of maintaining a therapeutic level of said protein for a sustained period of time, wherein the protein is present in an amount sufficient to protect the mammal for an extended period of time.

Particular embodiments of the present invention include a pharmaceutically acceptable dosage form, wherein the dosage form further comprises a component selected from the group consisting of a viscosity modifier and a surfactant.

The invention also relates to a method of preparing a pharmaceutically acceptable dosage form of a stabilized protein composition for parenteral administration to a mammal, the method comprising the step of combining a protein and a stabilizing buffer, the stabilized protein composition being capable of maintaining a therapeutic level of the protein for a sustained period of time, wherein the protein is present in an amount sufficient to protect the mammal for at least 3 days.

The present invention also relates to a method of treating or preventing an infection in a mammal, the method comprising administering to the mammal a stabilized protein composition comprising administering to the mammal a therapeutically effective amount of a stabilized protein composition, wherein the stabilized protein composition comprises a protein and a stabilizing buffer, the composition being capable of maintaining a therapeutic level of the protein for a sustained period of time.

Particular embodiments of the present invention include such methods of treating or preventing infection in a mammal, wherein the protein is G-CSF.

The invention also relates to a method of treating or preventing mastitis, metritis or bovine respiratory disease in a mammal comprising administering to the mammal a stable G-CSF composition comprising administering to the mammal a therapeutically effective amount of a stable G-CSF composition, wherein the stable G-CSF composition comprises G-CSF and a stabilizing buffer, the composition being capable of maintaining a therapeutic level of the protein for a sustained period of time.

The invention also relates to a method of maintaining a therapeutic level of a protein in a mammal for a sustained period, the method comprising administering to the mammal a stabilized protein composition, wherein the stabilized protein composition comprises the protein and a stabilizing buffer, the composition being capable of maintaining the therapeutic level of the protein for the sustained period.

Particular embodiments of the present invention include such methods for maintaining a therapeutic level of a protein in a mammal over a sustained period of time, wherein the stabilizing buffer is selected from the group consisting of HEPES, TES, and TRICINE.

Other embodiments of the present invention include such methods of maintaining a therapeutic level of a protein in a mammal over a sustained period, wherein the sustained period is at least about 3 days.

Other embodiments of the present invention include such methods for maintaining a therapeutic level of a protein in a mammal over a sustained period of time, wherein the protein is selected from the group consisting of a colony stimulating factor, a growth hormone, a cytokine, an antibody, and an antigen. Specific examples of cytokines include interleukins (e.g., interleukins 1-18) and interferons (e.g., interferons α, β, and γ).

More specific embodiments of the invention include such methods for maintaining a therapeutic level of a protein in a mammal over a sustained period of time, wherein the protein is a colony stimulating factor.

Other embodiments of the present invention include such methods for maintaining a therapeutic level of a protein in a mammal over a sustained period of time, wherein the protein is selected from the group consisting of human G-CSF, bovine G-CSF, and canine G-CSF.

Other embodiments of the invention include such methods for maintaining a therapeutic level of G-CSF in a mammal over a sustained period of time, wherein said G-CSF is present at a concentration of 0.01 to 5 mg/ml.

Other embodiments of the invention include such methods for maintaining a therapeutic level of G-CSF in a mammal over a sustained period of time, wherein the stabilizing buffer is selected from the group consisting of HEPES, TES and TRICINE.

Other embodiments of the invention include methods of maintaining a therapeutic level of G-CSF in a mammal over such extended periods of time wherein the concentration of the stabilizing buffer is from about 0.05M to about 2M.

Other embodiments of the present invention include such methods for maintaining a therapeutic level of G-CSF in a mammal over a sustained period of time, wherein said G-CSF is bovine G-CSF.

Other embodiments of the present invention include such methods for maintaining a therapeutic level of bG-CSF in a mammal over a sustained period of time, wherein the bG-CSF is at a concentration of 0.01mg/ml to 5 mg/ml.

Other embodiments of the invention include such methods for maintaining a therapeutic level of bG-CSF in a mammal over a sustained period of time, wherein the stabilizing buffer is selected from the group consisting of HEPES, TES and TRICINE.

Other embodiments of the invention include such methods for maintaining a therapeutic level of bG-CSF in a mammal over a sustained period of time, wherein the concentration of the stabilization buffer is from about 0.05M to about 2M.

The invention also relates to a kit for administering a stabilized protein composition to a mammal comprising a first container comprising a therapeutically effective amount of a protein and a second container comprising a pharmaceutically acceptable stabilizing buffer, wherein the therapeutically effective amount of the protein in the first container, when combined with the pharmaceutically acceptable stabilizing buffer in the second container, is capable of maintaining a therapeutic level of the protein in the mammal over a sustained period.

Particular embodiments of the invention include kits wherein the protein content is sufficient to protect a mammal for at least 3 days.

Preferred compositions of the invention are stable protein compositions comprising bovine G-CSF and HEPES buffer, which compositions are capable of maintaining the therapeutic levels of bovine G-CSF in a mammal in vivo for at least 3 days, wherein the compositions have a pH of about 7.5 and a temperature of about physiological temperature or 40 ℃. Such compositions are particularly useful when the mammal is a cow. More specifically, the HEPES buffer concentration is in the range of about 0.05M to about 2M. It is particularly preferred that the HEPES buffer concentration is about 1M, and preferably, the bovine G-CSF concentration is about 0.01 to 5 mg/ml. Most preferably, the bG-CSF concentration is about 0.1 mg/ml.

Preferably, the present invention also relates to a stabilized protein composition comprising bovine G-CSF and HEPES buffer, which composition is capable of ensuring an extended shelf life in the range of about 3 weeks to about 18 months. Particularly preferred wherein the HEPES buffer concentration is from about 0.05M to about 2M. Also preferred is a composition having a pH of about 7.5 and a temperature of less than about 40 deg.C, most preferably about 4 deg.C. Also particularly preferred is an extended shelf life of from about 6 months to about 1 year. Alternatively, such a shelf-life extended stable composition can maintain the temperature of the composition at about 40 ℃.

FIG. 1 shows a graph of stability (% recovery) as a function of time for a 0.1mg/ml bG-CSF solution (pH 7.5). Storage conditions were 0.1M, 1M and 2M HEPES buffer concentration at 40 ℃.

FIG. 2 shows a graph of stability (% recovery) as a function of time for a 0.1mg/ml bG-CSF solution (pH7.5) at 40 ℃ with TES buffer concentrations of 0.1M, 1M and 2M.

FIG. 3 shows a graph of stability (% recovery) as a function of time for a 0.1mg/ml bG-CSF solution (pH7.5) at 40 ℃ under storage conditions of 0.1M, 1M and 2M TRICINE buffer concentration.

FIG. 4 shows a graph of stability (% recovery) of a 2mg/ml bG-CSF solution (pH7.5) as a function of time at 40 ℃ under storage conditions of HEPES buffer concentrations of 0.1M, 1M and 2M.

FIG. 5 shows a graph of stability (% recovery) as a function of time for a 2mg/ml bG-CSF solution (pH7.5) at 40 ℃ with TES buffer concentrations of 0.1M, 1M and 2M.

FIG. 6 shows a graph of stability (% recovery) as a function of time for a 2mg/ml bG-CSF solution (pH7.5) at 40 ℃ under storage conditions of 0.1M, 1M and 2M TRICINE buffer concentration.

FIG. 7 shows the total PMNs (expressed as a percentage of the control, 0 hour value) of bovine peripheral blood treated with bG-CSF prepared in water, 1M HEPES buffer, 1M TES buffer, and 1M TRICINE buffer.

FIG. 8 is a graph showing the stability of bG-CSF (mg/ml concentration) dissolved in Neupogen as a function of time^*Buffer (control, pH4.0), HEPES buffer (pH7.4), PBS buffer (pH7.0), Hanks buffer (pH8.5), and bicarbonate buffer (pH 8.2).

FIG. 9 is a graph showing stability (% recovery) of bG-CSF (mg/ml concentration) in HEPES buffers of 1000mM, 500mM, 100mM, 50mM and 20mM as a function of time at 40 ℃.

FIG. 10 shows two thermograms (kcal/mole/deg.) of two bG-CSF solutions versus temperature (. degree.C.). The upper panel (highest temperature of 47 ℃) is bG-CSF prepared in PBS (pH7.5), and the lower panel (highest temperature of 59 ℃) is bG-CSF prepared in 1M HEPES (pH 7.5).

FIG. 11 shows a plot of bovine PMN (neutrophil)% versus time for three formulations: bG-CSF in water (control), bG-CSF in 1M HEPES, and bG-CSF in 1M HEPES + 10% polaxamer.

FIG. 12 shows the solubility of bG-CSF in 1M HEPES buffer (pH7.5), and the absorbance at 310nm as measured relative to the bG-CSF concentration (mg/ml).

FIG. 13 shows the stability (% of initial concentration) of bovine G-CSF in 1M HEPES buffer and PBS at 40 ℃.

FIG. 14 shows the stability (% of initial concentration) of human G-CSF in 1M HEPES buffer and PBS at 40 ℃.

FIG. 15 compares the stability (% of initial concentration) of human G-CSF and bovine G-CSF dissolved in 1M HEPES buffer (pH 7.5).

FIG. 16 shows the starting concentration percentage of bG-CSF over time (days) dissolved in 1M HEPES buffer (pH4.0) at 40 ℃.

FIG. 17 shows the starting concentration percentage of bG-CSF over time (days) in 1M HEPES buffer (pH7.5) at 40 ℃.

FIG. 18 shows the initial concentration percentage of bG-CSF over time (days) dissolved in 1M TES buffer (pH4.0) at 40 ℃.

FIG. 19 shows the initial concentration percentage of bG-CSF over time (days) dissolved in 1M TES buffer (pH7.5) at 40 ℃.

FIG. 20 shows RP HPLC results (% initial bG-CSF concentration) over time (weeks) for samples stored at 5 ℃.

FIG. 21 shows the results of SE HPLC (% initial bG-CSF concentration) of samples stored at 5 ℃ over time (weeks).

FIG. 22 shows RP HPLC results (% initial bG-CSF concentration) over time (weeks) for samples stored at 30 ℃.

FIG. 23 shows the results of SE HPLC (% initial bG-CSF concentration) of samples stored at 30 ℃ over time (weeks).

FIG. 24 shows RP HPLC results (% initial bG-CSF concentration) for samples stored at 40 ℃.

FIG. 25 shows the results of SE HPLC (% initial bG-CSF concentration) of samples stored at 40 ℃ over time (weeks).

FIG. 26 is a CD (circular dichroism) spectrum of bG-CSF.

FIG. 27 is a plot of molar ellipticity at a wavelength of 222nm as a function of temperature.

FIG. 28 shows the starting concentration percentage of bG-CSF over time (days) dissolved in HEPES buffer (pH7.5) at various concentrations at 40 ℃.

FIG. 29 is a graph of bG-CSF prepared in 1M HEPES versus time (in hours) post-injection versus WBC for the control formulation.

The present invention relates to stabilized protein compositions, and is based on the surprising discovery that: proteins, particularly proteins used in the treatment of mammalian (e.g., human, dog, cat, goat, sheep, horse and pig) infections, can be stabilized by adding a stabilizing buffer, such as HEPES, TES and TRICINE, to the protein so that the stabilized protein composition maintains protein activity for a sustained period of time (in vivo and in vitro). In terms of in vivo activity, the stabilized protein compositions of the invention are capable of maintaining these proteins at therapeutic levels in a mammal over a sustained period of time.

In particular, the present invention is a sustained release (sustained activity) formulation of bG-CSF dissolved in a stabilizing buffer (e.g., HEPES or TES) which provides prolonged pharmaceutical activity, bG-CSF is known to denature at around 40 ℃ and to be unstable at neutral pH. This is problematic because the physiological pH of cows is close to neutral and the body temperature is about 40 ℃.

The protein in the stabilizing composition of the present invention may be a naturally occurring protein, an isolated or purified protein, or a protein prepared by recombinant means. The invention also includes all chemically modified protein chemical modifications (e.g., methionine oxidation, cysteine S-alkylation and β -mercaptoethanol added disulfides, alkylation of lysine amino groups, etc.). The preferred protein for use in the stabilized protein compositions of the invention is G-CSF, most preferably bG-CSF.

G-CSF refers to granulocyte colony stimulating factor, including natural forms of granulocyte colony stimulating factor and variants and mutants, including recombinant variants having, for example, 1 or more amino acid deletions, substitutions and/or additions. These variants and mutants retain all or sufficient biological activity to be therapeutically effective in a mammal. The natural form of G-CSF is a glycoprotein which comprises a protein consisting of 174 amino acids and a form with three additional amino acids. Both forms have 5 cysteine residues, 4 form two disulfide bonds, and one is in free form.

Other examples of proteins suitable for use in the stabilized protein compositions of the invention include: for example, activins, adhesion molecules (e.g., L-selectin, CD-18, and ICAM-1), chemokines, erythropoietins, growth factors, statins, insulin, interferons (e.g., α, β, γ); interleukins (e.g., interleukins 1-18), leptin, macrophage inflammatory proteins, macrophage migration inhibitory factor, macrophage stimulating protein, neurotrophins, neutrophil inhibitory factor, oncostatin, somatostatin, growth hormone (of all species, e.g., porcine, bovine, or human), stem cell factor, tumor necrosis factor, thrombopoietin, as well as cell-associated and soluble receptors for all of the above proteins and any and all other proteins that provide a beneficial or therapeutic effect when administered to a mammal. Examples of specific proteins that can be used in the stabilized protein compositions of the invention are shown in Table 1. Other proteins that can be used in the stabilized protein compositions of the invention include those described in R & D Systems Catalogue (614 McKinley plant NE, Minneapolis MN 55413, USA) which are incorporated by reference.

TABLE 1

Potentially therapeutic proteins

β2-microglobulin (β)2M) 6-histidine 6Ckine Amphiregulin (AR) angiogenin(ANG) annexin VB-lymphocyte adhesion molecule (BL-CAM) beta endothelial cell growth factor (beta-ECGF) beta 0 nerve growth factor (beta-NGF) beta Actin (beta-Actin) beta animal cellulose (BTC) Brain Derived Neurotrophic Factor (BDNF) CD31(PECAM-1) CDIO ciliary neurotrophic factor (CNIF) ciliary neurotrophic factor receptor beta 1(CNTF R beta 2) CRG-2(IP-10) CXCR-1(IL-8 RA) CXCR-2(IL-8 RB) CXCR-3CXCR-4 (fusin) cytokine-induced neutrophil chemokine 1(CINC-1) cytokine-induced neutrophil chemokine 2 beta (CINC-2 beta) cytokine-induced neutrophil chemokine 2 alpha (CINC-2 alpha) cytotoxic T lymphocyte-associated molecule 4(CTLA-4) E-selectin endothelin 1(ET-1)

TABLE 1 (continuation)

Potentially therapeutic proteins

Eotaxin (eot) Eotaxin-2(Eot-2) Epidermal Growth Factor (EGF) epithelial derived neutrophil attractant 78(ENA-78) erythropoietin receptor (Epo R) erythropoietin (Epo) Fas (CD95) fibroblast growth factor 4(EGF-4) fibroblast growth factor 5(FGF-5) fibroblast growth factor 6(FGF-6) fibroblast growth factor 7/KGF (FGF-7) fibroblast growth factor 8(FGF-8) fibroblast growth factor 8b (FGF-8b) fibroblast growth factor 8c (FGF-8c) fibroblast growth factor 9(FGF-9) acidic fibroblast growth factor (acidic) basic fibroblast growth factor (basic FGF) Fibronectin (FN) Fit-1Fit-3 ligand Fractalkine glial cell line-derived neurotrophic factor (GDNF) glycoprotein Leukocyte 130(gp130) granulocyte chemotactic protein (GCP-2) granulocyte colony stimulating factor (G-CSF) granulocyte colony stimulating factor receptor (G-CSF R) granulocyte macrophage colony stimulating factor receptor (GM-CSF)

TABLE 1 (continuation)

Potentially therapeutic proteins

Growth-related protein (GRO) growth-related protein alpha (GRO alpha) growth-related protein alpha 0(GRO alpha 1) growth-related protein gamma (GRO gamma) hemofiltration CC chemokine I (HCC-1) heparin binding epidermal growth factor (HB-EGF) Hepatocyte Growth Factor (HGF) Heregulin alpha (HRG-alpha) Heregulin beta 1 (HRG-beta 1) I-309 insulin-like growth factor (IGF-1) Interleukin gamma (IFN gamma) Interleukin 1 receptor antagonist (IL-1ra) Interleukin 11 receptor (IL-11R) Interleukin 12p 70(IL-12 p70) Interleukin 13(IL-13) Interleukin 16(IL-16) Interleukin 2 receptor alpha (IL-2 Ra) Interleukin 2 receptor beta (IL-2R beta) Interleukin 3(IL-3) Interleukin 4 receptor (IL-4R) Interleukin 5(IL-5) Interleukin 7 receptor (IL-7R) Interleukin 7 Interleukin 9R (IL-9) IP-10JE/MCP-1 keratinocyte growth factor/FGF-7 (KGF)

TABLE 1 (continuation)

Potentially therapeutic proteins

L-selectin latency-related peptide (TGF-. beta.1) (LAP TGF-. beta.1) latent transforming growth factor beta 1 (latent TGF-. beta.1) Lepfln (OB) Leptin receptor (Leptin R) leukemia inhibitory factor receptor beta 0(LIF R. beta.1) Leukemia Inhibitory Factor (LIF) LFA-1 insulin-like growth factor I receptor (IGF-I R) insulin-like growth factor II (IGF-II) intercellular adhesion molecule 3(ICAM-3) intercellular adhesion molecule 1(ICAM-1) interleukin 11(IL-11) interleukin 1 receptor type I (IL-1R 1) interleukin 10(IL-10) interleukin 10 receptor (IL-10R) interleukin 12(IL-12) interleukin 12p40(IL-12p40) interleukin 13 receptor alpha (IL-13R. alpha) interleukin 15(IL-15) interleukin 17(IL-17) interleukin 18/1G1 (IL-1F) -18) interleukin 2(IL-2) interleukin 2 receptor gamma (IL-2 Rgamma) interleukin 3 receptor alpha (IL-3 Ralpha) interleukin 4(IL-4) interleukin 5 receptor alpha (IL-5 Ralpha)

TABLE 1 (continuation)

Potentially therapeutic proteins

Interleukin 6(IL-6) interleukin 6 receptor (IL-6R) interleukin 7(IL-7) interleukin 8(IL-8) interleukin 9 receptor (IL-9R) interleukin lalpha (IL-lalpha) interleukin lalpha 1 (IL-lalpha 2) interleukin I receptor type II (IL-RII) Mac-1 alpha 0 chain macrophage colony stimulating factor (M-CSF) macrophage colony stimulating factor receptor (M-CSF) macrophage inflammatory protein 1 gamma (MIP-1 gamma) macrophage inflammatory protein 2(MIP-2) macrophage inflammatory protein 3 alpha (MIP-3 alpha) macrophage inflammatory protein 3 beta (MIP-3 beta) macrophage inflammatory protein 1 alpha (MIP-1 alpha) macrophage inflammatory protein 1 beta (MIP-1 beta) macrophage Migration Inhibitory Factor (MIF) Macrophage Stimulating Protein (MSP) macrophage-derived chemotactic Chemokine (MDC/DC-CK1) MARC/MCP-3Midkine (MK) MIG monocyte chemotactic protein 1/MCAF (MCP-1) monocyte chemotactic protein 2(MCP-2) monocyte chemotactic protein 3(MCP-3) monocyte chemotactic protein 4(MCP-4)

TABLE 1 (continuation)

Potentially therapeutic proteins

Monocyte chemotactic protein 5(MCP-5) Neural Cell Adhesion Molecule (NCAM) neurotrophin 3(NT-3) neurotrophin 4(NT-4) tumor suppressor M (OSM) P-selectin (CD62P) placental growth factor (PIGF) placental growth factor 2(PIGF-2) plasma seleno glutathione peroxidase platelet GPIIb/GPIII a (CD41a) platelet derived endothelial cell growth factor (PD-ECGF) Platelet Derived Growth Factor (PDGF) platelet derived growth factor A chain (PDGF A chain) platelet derived growth factor AA (PDGF-AA) platelet derived growth factor AB (PDGF-AB) platelet derived growth factor B chain (PDGF B chain)) Platelet-derived growth factor BB (PDGF-BB) platelet-derived growth factor receptor alpha (PDGF R alpha) platelet-derived growth factor receptor beta (PDGF R beta) polytropic factor (PTN) Pre-B cell growth stimulating factor/SDF-1 (PBSF) RANTES Secretory Leukocyte Protease Inhibitor (SLPI) Stem Cell Factor Receptor (SCFR) Stem Cell Factor (SCF) stromal cell-derived factor 1 beta/PBSF (SDF-1 beta)

TABLE 1 (continuation)

Potentially therapeutic proteins

Stromal cell derived factor 1/PBSF (SDF-1) stromal cell derived factor 1 alpha/PBSF (SDF-1 alpha) thrombopoietin (Tpo) Thymus and Activation Regulatory Chemokine (TARC) Thymus Expression Chemokine (TECK) transforming growth factor alpha (TGF-alpha 2) transforming growth factor alpha 0 (TGF-alpha 1) transforming growth factor alpha 21.2 (TGF-alpha 31.2) transforming growth factor alpha 42 (TGF-alpha 52) transforming growth factor alpha 6 binding protein I (TGF-alpha 7bpI) transforming growth factor alpha 81 (TGF-alpha 91) transforming growth factor alpha 0 type II receptor (TGF-alpha 1RII) transforming growth factor alpha 4 type III receptor (TGF-alpha 5RIII) transforming growth factor/beta 5 (YGF-beta 5) transforming growth factor beta 3 (TGF-beta 3) TrkB tumor necrosis factor alpha 3 (TNF-alpha) tumor necrosis factor beta (TNF-beta) tumor necrosis factor Receptor type I (TNF RI) tumor necrosis factor type II receptor (TNF RII) vascular cell adhesion molecule 1(VCAM-1) Vascular Endothelial Growth Factor (VEGF)

Preferred proteins are those that can be used to treat or prevent infections in mammals such as humans, dogs, cows, pigs, goats, sheep, horses and cats. The infection may be a bacterial infection or a protozoan infection, or may be caused by a virus.

The term "infection" as used herein (unless otherwise specified) includes bacterial, protozoal, fungal and viral infections occurring in mammals, as well as diseases associated with such infections, which may be treated or prevented by administration of the stabilized protein compositions of the present invention.

Infectious diseases that can be treated with the stabilized protein compositions of the invention include, but are not limited to, bovine infectious diseases such as bovine mastitis associated with Staphylococcus aureus, Escherichia coli, Streptococcus uberis, Streptococcus dysgalactiae, Streptococcus agalactiae, Klebsiella, Corynebacterium, but are not limited to these bacteria; bovine respiratory disease associated with infectious bovine rhinotracheitis virus (IBR), parainfluenza virus (PI3), bovine viral diarrhea virus (BVD), Pasteurella haemolytica, Pasteurella multocida, and Haemophilus somnus; reproductive system diseases (e.g., metritis); bovine diarrhea associated with (but not limited to) E.coli and Eimeria sp.

Other infectious diseases that can be treated with the stabilized protein compositions of the invention include, but are not limited to, infectious diseases in dogs (e.g., pyoderma) and respiratory diseases in dogs, also known as kennel cough.

The stabilized protein compositions of the invention can also provide therapeutic effects in addition to the treatment or prevention of infection. An example of a therapeutic effect beyond the treatment or prevention of infection is the administration of recombinant human G-CSF to dogs and cats to improve the myelosuppression resulting from chemotherapy, thereby making them more amenable to cancer treatment.

The term "stable" as used herein (unless otherwise indicated) means that the therapeutic level of the protein is maintained over a sustained period. The maintenance of a therapeutic level of protein occurs after administration of the protein composition of the invention to a mammal, either before use in vitro or during storage. The stability of the protein composition of the invention can be determined by the percentage of the starting concentration with respect to time using the methods described herein.

The stabilized protein compositions of the invention maintain the protein at a therapeutic level after administration to a mammal such that the protein provides a therapeutic or beneficial effect over a sustained period of time. The term "sustained period" as used herein (unless otherwise specified) means a period of time during which a therapeutic level of protein is maintained following administration of a stabilized protein composition of the invention to a mammal, or alternatively prior to in vitro use or during storage.

The therapeutic level of the protein over a sustained period of time can ensure a longer period of improvement or therapeutic effect in the mammal than would be possible if the same protein were administered to the mammal without the stabilizing buffer, e.g., as compared to a control solution of the protein in water or PBS. Alternatively, under in vitro storage conditions, a sustained period of therapeutic levels of a protein can provide enhanced protein stability over a longer period of time than would be possible if the same protein were stored in the absence of a stabilizing buffer, e.g., as compared to a control solution of the protein in water or PBS). Preferably, the duration is at least 3 days. Most preferably, the duration is about 7 days or longer.

The term "therapeutic level" as used herein (unless otherwise indicated) means the amount of protein that produces a therapeutic effect in various administration therapies. This amount is readily determined by one skilled in the art. The amount of protein used will depend on the type and severity of the infection, the route of administration, etc.

By "stabilizing buffer" is meant any buffer which, upon binding to the protein in the stabilizing composition of the present invention, provides a stable protein composition which is capable of maintaining a therapeutic level of the protein over a sustained period of time. Maintenance of a therapeutic level can be determined by methods known in the art (e.g., measuring protein activity). Preferably, the stabilizing buffer operates at physiological pH. Stabilizing buffers include, but are not limited to, organic buffers (e.g., those zwitterionic buffers commonly referred to as "good buffers," which operate at a pH of 6 to 8.5). Examples of such stabilizing buffers include: HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid), TES (N-TRIS (hydroxymethyl) methyl-2-aminoethanesulfonic acid) and TRICINE (N-TRIS (hydroxymethyl) methylglycine), dicarbarsenic acid, bis (2-hydroxyethyl) -imino-TRIS (hydroxymethyl) methane (BISTRIS), piperazine-N, N' -bis (2-ethanesulfonic acid) (PIPES), imidazole, and TRIS (hydroxymethyl) aminomethane (TRIS). Examples of buffers that can be used in the stabilized protein compositions of the invention are shown in Table 2.

TABLE 2

Buffer solution			pKa
				MES	2- (N-morpholino) ethanesulfonic acid		5.96
bis-tris	Bis (2-hydroxyethyl) imino-tris (hydroxymethyl) methane		6.36
				ADA	N-2-acetamidoiminodiacetic acid		6.43
ACES	N- (2-acetamido) iminodiacetic acid		6.54
				PIPS	piperazine-N, N' -bis (2-ethane)Sulfonic acid)		6.66
MOPSO	3- (N-morpholine) -2-hydroxypropanesulfonic acid		6.75
				bis-tris propane	1, 3-bis [ tris (hydroxymethyl) methylamino]Propane		6.80
BES	N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid		6.88
				MOPS	3- (N-morpholine) propanesulfonic acid		7.01
TES	N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid		7.16
				HEPES	N-2-hydroxyethylpiperazine-N' -2-aminoethanesulfonic acid		7.31
DIPSO	3- [ N-bis (hydroxyethyl) -amino]-2-hydroxypropanesulfonic acid		7.35
				TAPSO	3- [ N- (Tris-hydroxymethyl) methylamino]-2-hydroxypropanesulfonic acid		7.39
POPSO	piperazine-N 'N' bis- (2-hydroxypropanesulfonic acid)		7.63
				HEPPSO	N-hydroxyethylpiperazine-N' -2-hydroxypropanesulfonic acid		7.73
Tricine	N-tris (hydroxymethyl) methylglycine		7.79
				EPPS	N-2-hydroxyethylpiperazine-N' -2-aminopropanesulfonic acid		8.00
Bicine	N, N-bis- (2-hydroxyethyl) glycine		8.04
				TAPS	N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid		8.11
AMPSO	3-N- (. alpha.,. alpha. -dimethylhydroxyethyl) -amino-2-hydroxypropanesulfonic acid		9.10
				CAPSO	3-N-Cyclohexylsulfamic acid		9.43

The pH of the stabilized protein compositions of the invention may range from about 4.0 to about 8.

The term "physiological pH" as used herein (unless otherwise specified) means the range of pH that exists in mammals, including humans, cows, pigs, horses, goats, sheep, dogs and cats. The physiological pH of mammals is typically in the range of about 6.5 to about 8.0.

The temperature of the stabilized protein compositions of the invention may range from about 20 ℃ to about 50 ℃.

The term "physiological temperature" as used herein (unless otherwise specified) means the range of body temperatures present in mammals, including humans, cows, pigs, horses, goats, sheep, dogs and cats. Physiological temperatures of mammals are generally in the range of about 37 ℃ to about 41 ℃. Physiological temperatures of some representative mammals are as follows: 37 ℃ for human body; cattle at 39 ℃; 38 ℃ for the cat; a dog at 39 ℃; the goat is at 39 ℃; horse 37 ℃; and 37 ℃ for pigs.

Preferably, the stabilized protein composition of the invention comprises protein G-CSF, more preferably bovine G-CSF in a stabilizing buffer selected from the group consisting of HEPES buffer, TES buffer and TRICINE buffer. The resulting stabilized protein composition is capable of maintaining a therapeutic level of bG-CSF activity for a duration of at least 3 days at a physiological pH of the bovine and at a physiological temperature of about 40℃ thereof.

The stabilized protein composition is prepared by combining the protein with a stabilizing buffer using known, commonly used binding techniques. Particular methods of preparing stable protein compositions include the use of proteins in purified form prepared according to protein purification techniques known to those skilled in the art.

For a particular protein of therapeutic value, the protein (at its maximum solubility) can be dissolved in various concentrations, such as from 0.05 to 2M of various buffers, e.g., HEPES, TES, TRICINE, or other buffers. In addition, the pH of the solution can vary, typically from about pH4.0 to about 8.0. The maximum solubility of a protein in a particular buffer can be determined by conventional means known in the art. The solution may then be stored at the physiological temperature of the mammal to which the protein solution is to be administered, and the amount of protein in the solution (as a function of time) determined. The therapeutic level of protein in solution can be determined by the% recovery of protein (as a function of time). The residual amount or% recovery of protein can be compared to a known threshold level required for the protein to produce a therapeutic effect. The number of days that the amount of protein remaining in solution is equal to or greater than a threshold level required for a known protein to produce a therapeutic effect can then be defined as the duration of time. A buffer that can be used as a stabilizing buffer is one that, when bound to a particular protein, maintains the therapeutic level of the protein for an extended period of time, i.e., a period of time longer than would be possible if the same protein were administered to a mammal in the absence of the stabilizing buffer.

Protein stability can be determined by measuring protein activity (as a function of time). Stretching temperature (T) of protein_m) Can be used as a marker of solution stability and stability of the protein in vivo. The stretching temperature of a particular protein is indicative of the temperature at which the protein loses secondary structure, and usually also loses activity, as can be determined by methods known to those skilled in the artSuch as differential scanning calorimetry.

The protein content of the stabilized protein compositions of the invention may be from about 0.1mg/ml to about 5 mg/ml. For G-CSF, the preferred range is from about 0.1mg/ml to about 3 mg/ml.

An example of a stabilized protein composition according to the present invention is a composition comprising bG-CSF and HEPES buffer, wherein the bG-CSF concentration ranges from about 0.1mg/ml to about 5mg/ml and the HEPES buffer concentration ranges from about 0.1M to about 2M. More preferably, the bG-CSF concentration ranges from about 0.1mg/ml to about 3 mg/ml.

Another example of a stabilized protein composition according to the invention is a composition comprising bG-CSF at a concentration in the range of about 0.1mg/ml to about 5mg/ml and TES buffer at a concentration in the range of about 0.1M to about 2M. More preferably, the bG-CSF concentration ranges from about 0.1mg/ml to about 3 mg/ml.

A further example of a stabilized protein composition according to the invention is a composition comprising bG-CSF at a concentration in the range of about 0.1mg/ml to about 5mg/ml and TRICINE buffer at a concentration in the range of about 0.1M to about 2M. More preferably, the bG-CSF concentration ranges from about 0.1mg/ml to about 3 mg/ml.

The stabilized protein compositions of the invention may be prepared in frozen or lyophilized form by conventional means known to those skilled in the art. The lyophilized form of the protein may be reconstituted with a stabilizing buffer. Alternatively, the solution is stored in liquid form for immediate use. Preferably, the stabilized protein compositions of the invention are in a liquid state, which retains activity over long periods of storage.

The stabilized protein compositions of the invention may be administered orally, parenterally (subcutaneous, intravascular, intraperitoneal and intramuscular), nasally (e.g., by inhalation), intraocularly or intradermally or by infusion methods in a manner known to those skilled in the art. Parenteral administration is preferred.

Regardless of the route of administration, the stabilized protein compositions of the invention may be formulated into pharmaceutically acceptable dosage forms by conventional methods known or apparent to those skilled in the art.

The stabilized protein compositions of the invention are preferably suitable for subcutaneous administration in a pharmaceutically acceptable dosage form. Pharmaceutically acceptable dosage forms for subcutaneous administration are generally no more than 20ml in volume (e.g., administered to horses and cattle), are sterile (suitable for use in mammals), and are well tolerated by mammals (i.e., do not induce swelling, pain or necrosis at the injection site).

In general, the pharmaceutically acceptable dosage forms of the invention may contain other pharmaceutically acceptable ingredients, such as surfactants or detergents, viscosity modifiers, sugars or proteins, in amounts suitable for effective, safe pharmaceutical administration. For example, the stabilized protein compositions of the present invention may be formulated into pharmaceutically acceptable dosage forms using carriers, stabilizers, diluents and/or preservatives as is conventional. Diluents may include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic additives may include sodium chloride, glucose, mannitol, sorbitol, lactose, and the like. The stabilizer may include albumin and the like. Other suitable carriers and additives will be known or apparent to those skilled in the art.

The stabilized protein compositions of the invention may be provided in the form of a kit comprising a first container containing a therapeutically effective amount of the protein and a second container containing a pharmaceutically acceptable stabilizing buffer. The protein may be in a solid state (e.g., frozen or lyophilized form) or a liquid state. The stabilization buffer can then be combined with the protein and administered to the mammal such that a therapeutically effective amount of the protein in a first container, when combined with a pharmaceutically acceptable stabilization buffer in a second container, is capable of maintaining a therapeutic level of the protein in the mammal for an extended period of time.

Particular embodiments of the invention include kits wherein the amount of protein is sufficient to protect the mammal for at least 3 days.

The pharmaceutically acceptable dosage form of the present invention may range from about 0.1. mu.g/kg to about 50. mu.g/kg, preferably from about 1. mu.g/kg to about 25. mu.g/kg, and most preferably from about 3. mu.g/kg to about 25. mu.g/kg. The most preferred dosage form for bG-CSF is about 24 μ g/kg. The dose is effective for at least about 3 days.

The following examples are intended to illustrate specific embodiments of the present invention, but the scope of the present invention is not limited to the examples described.

Example 1

Sustained stability of bG-CSF in HEPES, TES and TRICINE buffers

Three buffers were prepared at concentrations of 0.1M, 1M, 2M, respectively: HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid), TES (N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid) and TRICINE (N-tris (hydroxymethyl) methylglycine). Buffers were purchased from Fluka Biochemica USA. The pH of each buffer was adjusted to 7.5 with sodium hydroxide (j.t.baker, USA). The buffer was filter sterilized with a 0.2 μm GV filter (Millipore USA). The prepared buffer solution concentration was: HEPES buffer solution: 0.1M, 1M and 2M; TES buffer solution: 0.1M, 1M and 2M; TRICINE buffer: 0.1M, 1M and 2M.

Solutions containing 0.1mg/ml of G-CSF were prepared in various buffers (TES, TRICINE and HEPES) having the above-mentioned concentrations. The procedure was to add 4.69mg of bG-CSF (53.3% by titer) to a 25ml volume flask. The volume was then made up with the appropriate buffer concentration.

Solutions containing 2mg/ml bG-CSF were prepared in various buffers (TES, TRICINE, and HEPES) having the concentrations described in Table 1. The procedure was to add 93.8mg of bG-CSF (53.3% according to titer) to a 25ml volume flask. The volume was then made up with the appropriate buffer concentration.

The bG-CSF preparation was then filtered through a 0.22 μm low protein binding filter (Millipore USA). 1ml of each formulation was filled into a 1ml vial, which was then placed in an oven at 40 ℃ for 9 days. A stable bG-CSF buffer solution was prepared at 0.1mg/ml bG-CSF in (1) HEPES buffer: 0.1M, 1M and 2M; (2) TES buffer solution: 0.1M, 1M and 2M; and (3) TRICINE buffer: 0.1M, 1M and 2M. Samples were taken from each vial every 3 days for size exclusion HPLC (SEC-HPLC) analysis. The results are shown in FIGS. 1 to 6 and tables 3 and 4.

TABLE 3

	HEPES			TES			TRICINE
										Time (sky)	0.1M	1M	2M	0.1M	1M	2M	0.1M	1M	2M
0	10％	100％	100％	10％	100％	100％	100％	100％	10％
										3	15％	95％	78％	16％	85％	100％	17％	85％	94％
6	9％	96％	82％	11％	97％	98％	10％	79％	88％
										9	5％	95％	83％	9％	93％	98％	5％	70％	86％

Table 3 shows the recovery% (residual) of the 0.1mg/ml bG-CSF solution prepared as described above (function of time). The solution was stored at 40 ℃.

TABLE 4

	HEPES			TES			TRICINE
										Time (sky)	0.1M	1M	2M	0.1M	1M	2M	0.1M	1M	2M
0	100％	100％	100％	100％	100％	100％	100％	100％	100％
										3	2％	43％	62％	3％	78％	95％	2％	46％	72％
6	0％	37％	54％	2％	75％	89％	0％	38％	65％
										9	0％	33％	49％	0％	70％	88％	0％	31％	58％

Table 4 shows the recovery% (remaining) of the 2.0mg/ml bG-CSF solution prepared as described above (as a function of time). The solution was stored at 40 ℃.

FIGS. 1 to 3 show the stability under storage conditions at 40 ℃ of 0.1mg/ml bG-CSF solutions (pH7.5) buffered in HEPES, TES and TRICINE, respectively, at various concentrations (0.1M, 1M and 2M). As shown in FIGS. 1 to 3, the stability of bG-SF activity or residual amount was improved when the buffer concentration was increased to 1M and higher. The recovery of bG-CSF in 1M HEPES was 90% at 0.1mg/ml bG-CSF (FIG. 1).

FIGS. 4 to 6 show the stability under storage conditions at 40 ℃ of 2.0mg/ml bG-CSF solutions (pH7.5) dissolved in various concentrations (0.1M, 1M and 2M) of HEPES, TES and TRICINE buffers, respectively. As shown in FIGS. 4 to 6, when the buffer concentration was increased to 1M and higher, the stability of bG-CSF activity or the residual amount was also improved.

The data in tables 3 and 4 and figures 1 to 6 indicate that the presence of buffers (HEPES, TES and TRICINE) significantly maintained bG-CSF activity for a sustained period of 3 to 9 days.

Example 2

In vivo Effect of hG-CSF prepared in Water, 1M HEPES, 1M TES and 1M TRICINE buffer

Fruit

Cattle were taken for experiments to examine the in vivo effect of bG-CSF prepared in water, 1M HEPES, 1M TES and 1M TRICINE buffers. Cattle were dosed at 24 μ g/kg and the number of RMNs (neutrophils) was monitored.

FIG. 7 shows the total peripheral blood FMN (as a percentage of control, 0 hour value) of cattle treated with bG-CSF prepared in water, 1M HEPES, 1M TES and 1M TRICINE buffer. One injection, with all three buffers giving a recovery period of approximately 100 hours. This indicates that all three buffers delay protein activity at the injection site in vivo.

Example 3

Effect of HEPES buffer on the in vitro stability of bG-CSF

bG-CSF (described below) is formulated in various buffer systems to a concentration of 0.1mg/ml and a pH of about 7.0 to about 8.5. Before loading, all samples were filtered through a 0.2 micron filter (GV Millipore USA). The stable sample was packed into a column at 40 ℃ and monitored by reverse phase HPLC (rp HPLC), SEC HPLC and bioassay for 7 to 10 days. The stretching temperature of bG-CSF was measured using a VP-DSC MicroCalorimetry system (USA).

FIG. 8 shows the results of a comparison of the stability of bG-CSF in various buffer systems including Neupogen as a control^*(commercially available, USA, human G-CSF), HEPES (pH7.4), PBS (pH7.0), Hanks buffer (commercially available, USA), and bicarbonate buffer. The results indicate that bG-CSF formulated in 1M HEPES buffer is the most stable of all tested agents and shows a similarity to Neupogen^*Similar stability of the buffer (pH4.0). The stability of bG-CSF in HEPES buffer (shown in FIG. 8) was surprising and unexpected because bG-CSF was previously thought to be unstable at neutral or physiological pH and temperatures of about 40 ℃ or higher. bG-CSF prepared in PBS (pH7.0), Hanks buffer and bicarbonate buffer was unstable. Table 5 confirms this result.

TABLE 5

Buffer solution	Positive specific Activity (ng/ml) initiation	Specific activity positive (ng/ml)7 days (40 ℃ C.)*
			Neupogen*(pair)Light, pH4.0)	0.01	0.1
HEPES(pH7.4)	0.01-0.1	0.01-0.1
			PBS(pH7.0)	0.1	10
Hanks(pH6.4)	0.01-1.0	100
			Bicarbonate (pH8.2)	0.1-10	100-1000

^*Higher numbers correspond to lower activity

Dissolving in Neupogen^*And bG-CSF in HEPES buffer, which was not inactivated after being stored at 40 ℃ for 7 days. It was also shown that bG-CSF activity in PBS was 10-fold lower than the initial activity, while bG-CSF activity in Hanks and bicarbonate buffer was 100-1000-fold lower.

FIG. 9 shows the effect of HEPES buffer concentration (100mM, 50mM, and 20mM) on bG-CSF stability in HEPES at 40 ℃. As shown in FIG. 9, the stability of bG-CSF decreased significantly as the HEPES concentration decreased.

FIG. 10 is a thermogram of two different bG-CSF solutions. The upper thermogram (max. temperature 47 ℃ C.) was bG-CSF prepared in PBS (pH7.5), and the lower thermogram (max. temperature 59 ℃ C.) was bG-CSF prepared in 1MHEPES (pH 7.5). Without HEPES buffer, bG-CSF stretches at about 40 deg.C (starting temperature) at pH7.5, whereas bG-CSF stretches in 1M HEPES increase by 10 deg.C. An increase in the stretching temperature represents more stability.

Example 4

In vivo Effect of bG-CSF prepared in 1M HEPES

Cattle were taken for experiments to examine the in vivo effect of bG-CSF prepared in 1M HEPES. Cattle were dosed at 12 μ g/kg and the number of White Blood Cells (WBC) and PMN (neutrophils) was monitored. The results are shown in FIG. 11.

Fig. 11 is a graph of PMN% (neutrophils) as a function of time comparing three formulations: bG-CSF in water (as control), 1M HEPES + 10% polaxamer. As shown in fig. 11, the number of PMNs stayed above the threshold (the level associated with protection) for 3 days or 72 hours. 6 cattle were tested for each formulation.

In a second study, cattle were dosed at 24 μ g/kg with bG-CSF in 1M HEPES + 10% polaxamer, giving approximately 200 hours of protection, or nearly 8 days of recovery, in one injection. This result shows that HEPES buffer improves the in vivo stability of bG-CSF, thereby extending its activity maintenance period and protein delivery period.

Example 5

Solubility of bG-CSF in 1M HEPES

The solubility of bG-CSF in 1M HEPES (pH7.5) was determined. Approximately 80mg of bG-CSF was dissolved in 30ml of 1M HEPES buffer (pH 7.5). The protein solution was filtered through a 0.2 μm GV Millipore filter and then transferred to a 50ml ultrafilter. The cell was equipped with a low protein binding membrane with a molecular weight cut-off (MW) of 10000. The protein solution was concentrated using an ultrafiltration cell. At various times, samples were taken from the cell at 310nm for UV-Vis analysis (measurement of light scattering) and concentrated by RP HPLC. The absorbance at 310nm was plotted against concentration. The absorbance at 310nm increased linearly with concentration; the 310nm curve abruptly changes at the saturation concentration, and the absorbance sharply increases. The concentration at this point is the maximum solubility. Such methods are known to those skilled in the art and are commonly used to determine protein solubility. As shown in FIG. 12, the maximum solubility of bG-CSF in 1M HEPES (pH7.5) was about 5 mg/ml. The concentration at the curve mutation represents the maximum solubility of the protein. At a concentration of about 5mg/ml, the absorbance at 310nm rises abruptly, corresponding to the maximum solubility of the protein.

Example 6

Effect of HEPES, TES and TRICINE buffers on bG-CSF extension temperature

Preparing a solution containing 0.5mg/ml of G-CSF in 1M HEPES, 2M HEPES, 1M TES, 2M TES and 1M TRICINE; and a solution containing 2mg/ml bG-CSF in 1M HEPES, 2M HEPES, 1M TES, 2M TES and 1M TRICINE. These solutions were prepared in the same manner as described in example 1. A control solution was prepared with PBS (Dulbecco's phosphate buffered saline, pH 7.4). The pH of the bG-CSF solution was 7.5. The bG-CSF extension temperature was measured using differential scanning calorimetry (Microcal Inc. USA) at a scan rate of 60 degrees per hour over a temperature range of 20 ℃ to 90 ℃. The results are shown in Table 6.

TABLE 6

Stretching temperature (. degree.C.)

	PBS	HEPES		TES		TRICINE
							bG-CSF concentration (mg/ml)		1M	2M	1M	2M	1M
0.5	50.96	56.85	60.05	57.29	62.37
							2		54.94	57.62	55.23	60.49	53.68

The extension temperature is an indicator of solution stability as well as the stability of the protein in vivo. The results in Table 6 show the stretching temperature (T) of bG-CSF dissolved in HEPES, TES or TRICINE buffer at 1M and higher concentration_m) Significantly higher than the PBS control. Three kinds of slow solutions make T_mThe temperature rises by about 2 to 11 c. The concentration of the buffer greatly influences T_mThe degree of elevation. T of bG-CSF_mIncreasing with increasing buffer concentration. When the HEPES concentration was increased from 1M to 2M, the increase was about 3 ℃, and when the TES concentration was increased from 1M to 2M, the increase was about 5 ℃. And when the bG-CSF concentration increases, its T_mAnd (4) descending. The bG-CSF concentration in HEPES and TES decreased by about 2 ℃ when increased from 0.5mg/ml to 2 mg/ml. TES buffer solution for allowing T of bG-CSF to be equal to or higher than T of bG-CSF at a buffer solution concentration of 2M, bG-CSF of 0.5m/ml_mThe temperature is increased by more than 11 ℃.

The results in Table 6 show that all three buffers (HEPES, TES and TRICINE) cause T of bG-CSF in comparison with PBS_mIs remarkably increased. The stability of bG-CSF solution in 2M TES was highest compared to other buffers.

Example 7

Comparison of the stability of human and bovine G-CSF formulations in PBS and HEPES

0.15mg/ml hG-CSF and bG-CSF preparations were prepared in phosphate buffer (Dulbecco's PBS, pH7.4) and 1M HEPES buffer (1M HEPES, pH 7.5). The preparation was filled in a 1ml bottle (capacity 400ml) and stored at 40 ℃ for 10 days. Samples were checked every 3 days by size exclusion chromatography (SEC-HPLC) and visual inspection.

Figure 13 shows that human G-CSF dissolved in 1M HEPES buffer has significantly improved stability compared to PBS. Human G-CSF degrades upon storage at 40 ℃ for 10 days when prepared in PBS (pH7.4), whereas a recovery of 65% is observed when prepared in 1M HEPES buffer.

FIGS. 13 and 14 show that bovine G-CSF is slightly more stable in HEPES and PBS formulations than human G-CSF. The recovery of the bovine G-CSF preparation dissolved in 1M HEPES after 10 days storage at 40 ℃ was approximately 80%, whereas the human G-CSF was 65%. Both proteins (human and bovine G-CSF) were much more stable in 1M HEPES buffer than in PBS.

Example 8

Stability of human and bovine G-CSF in 1M HEPES formulations

A preparation of 0.1mg/ml hG-CSF and bG-CSF was prepared in 1M HEPES buffer (1M HEPES, pH 7.5). The preparation was filled into a 1ml bottle (capacity 400. mu.l) and stored at 40 ℃ for 10 days. Samples were checked every 3 days by size exclusion chromatography (SEC-HPLC) and visual inspection.

FIG. 15 shows the stability of bovine G-CSF and human G-CSF in 1M HEPES formulations. After 10 days of storage at 40℃, recovery of bovine G-CSF was about 90% and recovery of human G-CSF was about 70%.

Example 9

Effect of pH of the formulation on bG-CSF stability

A preparation of 0.1mg/ml bG-CSF solution was prepared in 1M HEPES and 1M TES buffers pH4.0 and pH 7.5. The preparation was filled into a 1ml bottle (capacity 400. mu.l) and stored at 40 ℃ for 10 days. Samples were checked every 3 days by size exclusion chromatography (SEC-HPLC).

As shown in FIGS. 16 and 18, the recovery of bG-CSF after 10 days of storage at 40 ℃ is about 100% at pH4.0, while FIGS. 17 and 19 show that the recovery is about 80-85% for the bG-CSF formulation at pH 7.5.

Example 10

Long-term thermal stability study of bG-CSF 6 month samples dissolved in 1.0M HEPES and TES formulations

The formulations included in this study are given below. Commercial 1.0M HEPES preparation from GibcoBRL (Lot #1016436), 1.0M Tes was prepared with powder from Fluka Scientific (Lot # PA 12602). The buffer pH was adjusted to 7.5. BG-CSF (53.3% purity) was supplied by Bioprocess (Lot # BP 185-11).

0.1mg/ml and 2.0mg/ml bG-CSF formulations were prepared in 1.0M HEPES and 1.0M TES buffer.

Sample storage A3.5 ml Flint Type 1 bottle (Lot # R04105-7322) with a 13mm 1888 Gray T/F stopper (Lot # R05619-7487) was used with a 1.0ml volume. Table 7 gives the total sample storage and sampling points. Each formulation was stored in 5 vials for each test time point.

Table 7 sample storage and titer test time point summary

Storage of	Initiation of	3 weeks	6 weeks	For 12 weeks	6 months old	1 year	18 months old
								5℃	×	×	×	×	×	×	×
30℃		×	×	×	×	×	×
								40℃		×	×	×	×

The 2.0mg/ml bG-CSF preparation was diluted 10-fold before HPLC analysis.

3 samples (5, 30, 40 ℃) of each formulation were removed from each reservoir and assayed for bG-CSF potency by RP and SEHPLC. Each sample was measured 3 times. The concentration was calculated using a pre-drawn standard curve. The percent (%) of the initial bG-CSF concentration of each test sample was determined and the mean value of each formulation at each time point was calculated. The% mean of the initial bG-CSF concentration at each storage temperature is plotted against time, indicating the decrease in bG-CSF titer. FIGS. 20 and 21 are the results of RP and SE HPLC, respectively (samples stored at 5 ℃ C.). FIGS. 22 and 23 show the results of sample storage at 30 ℃ and the results of sample storage at 40 ℃ are shown in FIGS. 24 and 25.

Except for the 0.1mg/ml protein preparation dissolved in 1M TES, there was little degradation of bG-CSF in samples stored at 5 and 30 ℃.

Example 11

Stabilization of biotherapeutic proteins by HEPES buffer

Determination of the T of the proteins discussed below in phosphate and HEPES buffers Using a MicroCal (VP-DSC type) microcalorimeter_mThe value is obtained. Using Na from Aldrich₂HPO₄(Lot #08019PQ) 25mM phosphate buffer was prepared and pH adjusted to 7.5. 1.0M HEPES buffer was purchased from GibcoBRL (pH7.5, Lot # 1016436). With stirred ultrafiltrating filters (model 8010, Amicon Inc.), using YM10 or YM30 Diaflo in combination^*Ultrafiltration membranes (Amicon Inc.) were buffer exchanged (depending on protein size).

2.0mg of lyophilized pST (Lot #41509-217-2) from Bioprocess were reconstituted in 2.0ml of Milli-Q water. After 5 exchanges, 1.0ml of reconstituted protein was transferred to 25mM phosphate buffer using YM10 membrane. The remaining 1.0ml was exchanged into 1.0M HEPES buffer. Samples of 1.0gm/ml protein concentration were prepared and then subjected to micro-calorimetry. Microcalorimetry of pST was performed twice in both phosphate and HEPES buffers.

Approximately 2.0ml of NIF (Lot #440631-22-7) at a concentration of 2.97 was purchased from Bioprocess. Samples from Bioprocess were divided into two. 1.0ml of the protein was exchanged into 25mM phosphate buffer, and the remaining NIF was exchanged into 1.0M HEPES buffer. In each case, the membrane was exchanged 5 times with YM 30. A solution was prepared at a concentration of 1.0mg/ml in the appropriate buffer. Determination of T by microcalorimetry_mThe value is obtained.

Biological therapeutic proteins detected and respective T's measured in phosphate and HEPES buffers_mThe values are given in Table 8.

TABLE 8 four biotherapeutic proteins obtained by microcalorimetry in phosphate and HEPES buffers

T of_mValue of

Protein	T in phosphatem	T in HEPESm	Tm
				NIF	59.79	63.27	+3.48
PST	(1)56.14	67.90	+11.76
					(2)54.48	63.41	+8.93

Table 8 shows that HEPES buffer improved the stability of both NIF, pST.

Example 12

Extension of the in vivo Activity of recombinant bovine granulocyte colony stimulating factor (rbG-CSF) with HEPES buffer

Bovine granulocyte colony stimulating factor (bG-CSF) was obtained from Bioprocess Research and development-Pfizer (Groton, CT), mannitol was obtained from E.M. industries (Hawthorne, NY), 1 × Dulbecco's Phosphate Buffered Saline (PBS) was obtained from GibcoBRL (Grandis land, NY), sodium citrate and sodium acetate were obtained from Aldrich (Milwaukee, WI), Tween-80, sodium chloride and hydrochloric acid were obtained from J.T. Baker (Phillipsburg, USA).

RP-HPLC Size Exclusion Chromatography (SEC)

The stability of the solution was monitored by RP-HPLC and SEC. RP-HPLC was performed using Vydac Protein C4 column, mobile phase: 0.1% TFA H2O (solvent a) and 0.1% TFA CAN (solvent B); flow rate: 1 ml/min; and (4) UV monitoring: 220 nm; temperature: at 25 ℃. With TosoHaas, TSK-GEL SW_XL(7.8mm ID. times.30 cm) column for size exclusion chromatography using mobile phase: 0.3M NaCl in 0.0SM citrate buffer (pH 5.75); flow rate: 1 ml/min; and (4) UV monitoring: 280 nm: temperature: at 25 ℃.

Microcalorimetry detection

Denaturation Temperature (TD) was measured with VP DSC system (MicroCal, Inc.). About 1ml of the solution was added to the cell at about 10 deg.C/min. Is performed relative to a reference blank formulation.

Circular dichroism

Using circular dichroism spectrophotometers (CD) equipped with temperature scanning measuring accessories^*ORD J-710/720 type-Japan Spectroscopic Co., LTD) monitors the secondary structure of bG-CSF.

Biological assay

The in vitro activity of the bG-CSF preparation was determined using the murine bone marrow cell proliferation assay (BMC method). Bone marrow cells were collected aseptically from femurs of female CF1 mice (Charles River) by removing the femurs and gently flushing bone marrow from the bones using a 3cc/23G syringe and Hanks Balanced salt solution (Gibco BRL). Thin and thinThe cell suspension was filtered through a nylon sieve to remove the residue, and centrifuged at 1100rpm for 10 minutes at room temperature. The supernatant was discarded and the pellet was resuspended in 15ml of RPM1 medium (Gibco BRL) supplemented with 10% fetal bovine serum (Gibco BRL), 1% streptomycin penicillium (10000 units/ml), 1% L-glutamine (Gibco BRQ). The number of bone marrow cells was measured by Coulter Channelyzer256, and the cell concentration was adjusted to 6.67X 10⁵Add about 10 cells/ml to each well of a 96 well plate⁵A cell. Different concentrations of bovine granulocyte colony stimulating factor (in triplicate) were then added to each well. After incubation at 37 ℃ for 3 days, in 5% CO₂The temperature is kept for 3 days. Adding to each well³H-thymidine (New England Nuclear, Boston, Mass) to a final concentration of 2. mu. Ci/ml. Radiolabelling at 37 ℃, (5% CO)₂) For at least 18 hours. Plates were frozen at-20 ℃ and after thawing the cells were collected into glass 96 well fiber discs using a Brandel cell collector (biological Research and development laboratories, Gaithersburg, Maryland). Activity was measured with a Wallac 1205 Betaplate liquid scintillation counter (Wallac Gaithersburg, Maryland). The activity of the bG-CSF preparation was determined by dividing the sample counts per minute by the vehicle control counts per minute (fold over background). Positive was taken to be 3 or more times background.

bG-CSF Activity in vivo

The in vivo activity of bG-CSF formulations was tested in young, hybrid cattle (body weights in the range of about 100-150 kg). The purchased cattle were shipped to an animal health research center (Terre Haute, Indiana) and allowed to acclimate to the apparatus for at least two days prior to the experiment. Most cattle were used in one experiment, allowed to rest for at least one week, and then a second experiment was performed. No cattle were used for more than two experiments. Prior to entering an experiment, cattle were prescreened by assessing rectal temperature, weight, constitution and total White Blood Cell (WBC) count and classification 1-3 days before the start of the experiment. Usually the rectal temperature is more than or equal to 104 ℃ and the total WBC number (4000/mm)³> or 12000/mm³Cattle of (4) were excluded. At day 0, bovine blood was taken and weighed before treatment. The dose of bG-CSF preparation (24. mu.g/kg) administered per cow over the treatment period was calculated based on body weight and was administered neck-wiseThe medicine is injected subcutaneously at the anterior shoulder blade. After treatment, blood samples were collected by venipuncture into EDTA anticoagulant at the neck at predetermined times for WBC/differential counting. Total WBC were counted in a NovaCelltrak I hemocytometer with whole blood diluted 1: 250 in an isotonic diluent. Differential WBC counts were performed using dried blood smears stained with a Diff-Quik staining apparatus (Dade). 100 WBCs were counted and classified on a Zeiss optical microscope with 100 x oil lens and 12.5 x eyepiece (total magnification 1250 x).

Effect of pH and temperature on bG-CSF solution stability

Table 9 shows the effect of temperature on bG-CSF stability. After influencing the stability of the bG-CSF solution with temperature, RP-HPLC, SEC-HPLC, biological detection and visual inspection were carried out (formulation: 0.1mg/ml bG-CSF, 5% mannitol, 10mM acetate buffer, 0.004% Tween-80, pH 4.0). It can be seen from Table 9 that the stability of bG-CSF is interrupted at 40 ℃ or higher. In both RP-HPLC and SEC-HPLC, the parent protein peak was lost at 40 ℃ and higher. At higher temperatures, the parent protein peak disappears, with an increase in microparticles in solution. This result was observed both visually and by light scattering monitoring at 310 nm. Bovine G-CSF solution stored at 40 ℃ for 3 weeks is 10-100 times less active than when stored at 5 ℃ and 30 ℃. After 3 weeks of storage at 50 ℃ bG-CSF is 100-fold lower than that of solutions stored at 5 ℃ and 30 ℃.

TABLE 9Stability of bG-CSF vs. temperature (3 weeks stability)

Temperature (. degree.C.)	RP-HPLC (% of Start)	SEC-HPL (% of Start)	Tyndall BeamVisual inspection results of (1) (310nm light scattering Abs.)	Specific activity positive (ng/ml, BMC test)*
					5	88.5	97.6	Limpid (0.2au)	0.1-1
30	67.3	76.8	Limpid (0.04au)	0.1-1
					40	5.6	5.4	Slightly turbid (> 0.1au)	10-100
50	1.9	No parent peak	Turbid (> 0.1au)	100-1000

^*Activity detected in murine BMC (note: high values indicate low activity)

The effect of temperature on bG-CSF was followed by Circular Dichroism (CD). The CD spectrum of bG-CSF is shown in FIG. 26. This spectrum suggests that the secondary structure of bG-CSF is mainly an α -helix, and is structurally very similar to human G-CSF. CD spectra were measured at different temperatures to determine the denaturation Temperature (TD). FIG. 27 is a graph of molar ellipticity at a wavelength of 222nm (characteristic wavelength of. alpha. -helix) versus temperature. Between 40-50 ℃, an increase in molar ellipticity indicates a loss of secondary structure and denaturation of bG-CSF.

The effect of pH on the stability of bG-CSF solutions was also tested. To describe only the effect of pH on stability, not the effect of temperature-induced denaturation, the solution was stored at 30 deg.C (TD of bG-CSF between 40-50 deg.C). Table 10 summarizes the relationship between bG-CSF stability and pH during storage at 30 ℃ for 2 weeks. The rate of loss of protein activity increases with increasing pH. The data indicate that at low pH, the cysteine in bG-CSF is protonated and therefore the formulation is more stable. At high pH, the free cysteine participates in the disulfide exchange reaction, a possible cause of instability.

Watch 10Relation between bG-CSF stability and pH (stability after storage at 30 ℃ for 2 weeks)

pH	RP-HPLC (% of Start)	SEC-HPLC (% of Start)	Visual inspection of Tyndall Beam	Specific activity positive (ng/ml, BMC test)*
					4.0	96.5	100	Clear solution	1-10
5.0	91.9	90.1	Clear solution	10-100
					6.0	75.2	84.6	Fine particles	100
7.0	30.5	45.7	Long gel particles	100-1000

^*Activity detected in the murine BMC assay (note: higher values represent lower activity)

Effect of HEPES buffer on bG-CSF solution stability

We observed that the bG-CSF formulation dissolved in 1M HEPES buffer showed higher solution stability even when stored at 40 ℃ for several days. This is unexpected because bG-CSF is known to be unstable at neutral pH and denature at 40 ℃ or higher. FIG. 28 shows the effect of HEPES buffer concentration on bG-CSF solution stability at storage temperature of 40 ℃ and pH 7.5. The stability of bG-CSF decreases significantly as the HEPES buffer concentration decreases. The effect of 1M HEPES on the denaturation Temperature (TD) of bG-CSF was determined by microcalorimetry. FIG. 10 (thermogram) compares the effect of two formulations (with and without 1M HEPES) on TD of bG-CSF. Without HEPES buffer, the endothermic transition was initiated at about 40 ℃ while the TD onset of bG-CSF dissolved in 1M HEPES was around 50 ℃. An increase in the denaturation temperature is often a sign of stabilization.

In vivo Activity of bG-CSF

We evaluated the in vivo activity of this formulation in cows. FIG. 29 is a WBC count graph over time comparing bG-CSF prepared in 1M HEPES with a "control" formulation. "control" is a formulation containing 5% mannitol, 10mM acetate buffer, Tween-80 (pH4.0).

As can be seen in fig. 29, the WBC count stayed above the threshold (level that prevented infection) only for approximately 24-30 hours (200% baseline level). However, when bG-CSF is prepared in 1M HEPES, the number of PMNs stays above the threshold for a minimum of 3 days or 72 hours (in some cases the WTC stays above the threshold for a week). This experiment was reproducible (6 cattle per formulation in each experiment). The experimental results suggest that HEPES buffer can not only be used as an in vitro stabilizer, but also improve the in vivo effect of bG-CSF in some way.

The unexpected finding that HEPES buffer affects the effect of bG-CSF prompted us to investigate similar buffers, such as MOPS, HEPPS, TES, and TRICINE. These buffers, like HEPES, also improve the in vitro stability of bG-CSF. In vivo experiments were performed on bG-CSF dissolved in TES and TRICINE buffers, and both preparations extended the activity of bG-CSF in cows, similar to HEPES preparations.

The dissolution of bG-CSF in 1M HEPES prolonged the activity of bG-CSF in vivo. This prolonged activity may be the result of increased bG-CSF stability at the injection site. When bG-CSF is prepared in 1M HEPES, the solution stability of bG-CSF is significantly improved at neutral pH and at a temperature of 40 ℃. Other organic buffers (e.g., MOPS, HEPPS, TES, and TRICINE) can also provide enhanced bG-CSF stability.

Claims (22)

1. A stable composition comprising a granulocyte colony stimulating factor and an organic stabilizing buffer selected from the group consisting of HEPES, TES and TRICINE, said composition being capable of maintaining a therapeutic level of the granulocyte colony stimulating factor for an extended period of time.

2. The composition according to claim 1, wherein the granulocyte colony stimulating factor is selected from the group consisting of human G-CSF, bovine G-CSF and canine G-CSF.

3. The composition according to claim 2, wherein the granulocyte colony stimulating factor is bovine G-CSF.

4. The composition according to claim 1 or 3, wherein the composition is at a pH range of about 6.5 to about 8.0.

5. The composition according to claim 1 or 3, wherein the composition is at a pH range of about 4.0 to about 7.5.

6. The composition according to claim 1 or 3, wherein the composition is at a temperature of from about 37 ℃ to about 41 ℃.

7. A composition according to claim 2 or 3, wherein the concentration of G-CSF is from 0.1mg/ml to 5 mg/ml.

8. The composition according to claim 1, 2 or 3, wherein the concentration of the organic stabilizing buffer is from about 0.1M to about 2M.

9. The composition according to claim 3, wherein the organic stabilizing buffer is HEPES at a concentration of about 1M.

10. A pharmaceutically acceptable dosage form of a stabilized protein composition for parenteral administration to a mammal comprising a granulocyte colony stimulating factor and a pharmaceutically acceptable organic stabilizing buffer, wherein said organic stabilizing buffer is selected from the group consisting of HEPES, TES and TRICINE, said composition being capable of maintaining a therapeutic level of said granulocyte colony stimulating factor for a time period, wherein said granulocyte colony stimulating factor is present in an amount sufficient to provide a therapeutic effect to the mammal for a predetermined period of time.

11. The pharmaceutically acceptable dosage form according to claim 10, wherein the dosage form further comprises a component selected from the group consisting of a viscosity modifier and a surfactant.

12. The pharmaceutically acceptable dosage form according to claim 10, wherein the granulocyte colony stimulating factor is bovine G-CSF present in a concentration of about 0.1mg/ml to 5mg/ml, the mammal is a dairy cow, the predetermined period of time is at least about 3 days, and the composition has a pH of about 7.5.

13. The pharmaceutically acceptable dosage form according to claim 12, wherein the organic stabilizing buffer is HEPES at a concentration of about 0.1M to about 2M.

14. Use of a stable protein composition comprising granulocyte colony stimulating factor and an organic stabilizing buffer, wherein the organic stabilizing buffer is selected from the group consisting of HEPES, TES and TRICINE, in the manufacture of a medicament for treating or preventing infection in a mammal, the composition being capable of maintaining a therapeutic level of the granulocyte colony stimulating factor for a duration of at least about 3 days.

15. Use according to claim 14, wherein the mammalian infection is bovine mastitis, metritis or bovine respiratory disease.

16. Use of a stable granulocyte colony stimulating factor composition comprising a granulocyte colony stimulating factor and an organic stabilizing buffer, wherein the organic stabilizing buffer is selected from the group consisting of HEPES, TES and TRICINE, in the manufacture of a medicament for maintaining a therapeutic level of granulocyte colony stimulating factor in a mammal for a sustained period, wherein the composition is capable of maintaining the therapeutic level of granulocyte colony stimulating factor for a sustained period of about at least 3 days.

17. The method or use according to claim 14, 15 or 16 wherein the granulocyte colony-stimulating factor is about 0.1mg/ml to 5mg/ml of bovine G-CSF and the composition has a pH of about 7.5.

18. A kit for administering a stable granulocyte colony stimulating factor composition to a mammal comprising a first container comprising a therapeutically effective amount of granulocyte colony stimulating factor and a second container comprising a pharmaceutically acceptable organic stabilizing buffer selected from the group consisting of HEPES, TES and TRICINE, wherein the therapeutically effective amount of granulocyte colony stimulating factor in the first container when combined with the pharmaceutically acceptable organic stabilizing buffer in the second container maintains the therapeutic level of the granulocyte colony stimulating factor in the mammal for a sustained period of at least about 3 days.

19. The kit according to claim 18, wherein said granulocyte colony-stimulating factor is bovine G-CSF in a concentration range of about 0.1mg/ml to 5mg/ml and said composition has a pH of about 7.5.

20. A stable protein composition comprising bovine G-CSF and HEPES buffer which provides an extended shelf life in the range of about 3 weeks to about 18 months.

21. The composition according to claim 20, wherein the HEPES buffer is at a concentration of about 0.1M to about 2M, the composition has a pH of about 7.5, and the composition has a temperature of less than about 40 ℃.

22. The composition according to claim 21, wherein said temperature is about 4 ℃.