Lyophilized formulations of antibacterial proteins

Abstract

A freeze-dried formulation includes an antibacterial protein having a killing activity specific to at least one or all of the following genera: Staphylococcus arlettae, Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus carnosus, Staphylococcus caprae, Staphylococcus chromogenes, Staphylococcus cohnii, Staphylococcus delphini, Staphylococcus epidermidis, Staphylococcus equorum, Staphylococcus gallinarum, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus intermedius, Staphylococcus kloosii, Staphylococcus lentus, Staphylococcus lugdunensis, Staphylococcus muscae, Staphylococcus pasteuri, Staphylococcus saprophyticus, Staphylococcus warneri, and Staphylococcus xylosus; a poloxamer; a sugar; and an amino acid.

Description

[0001] Related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 277,588, filed January 12, 2016, which is incorporated herein by reference for all purposes as if fully set forth herein. Technical Field

[0003] This invention relates to antimicrobial proteins, specifically freeze-dried formulations of antimicrobial proteins specific to at least one or all of the following genera: *Staphylococcus arlettae*, *Staphylococcus aureus*, *Staphylococcus auricularis*, *Staphylococcus carnosus*, *Staphylococcus carprae*, *Staphylococcus chromogenes*, *Staphylococcus cohnii*, *Staphylococcus delphini*, *Staphylococcus epidermidis*, *Staphylococcus equorum*, *Staphylococcus gallinarum*, *Staphylococcus hemolyticus*, *Staphylococcus hominis*, and *Staphylococcus intermedius*. The following species are listed: *Staphylococcus intermedius*, *Staphylococcus kloosii*, *Staphylococcus lentus*, *Staphylococcus lugdunensis*, *Staphylococcus muscae*, *Staphylococcus pasteuri*, *Staphylococcus saprophyticus*, *Staphylococcus warneri*, and *Staphylococcus xylosus*. Background Technology

[0004] A bacteriophage is any of a variety of virus-like microorganisms that infect bacteria, and the term is usually used in its abbreviation, "phage." A bacteriophage with specific bactericidal activity against Staphylococcus aureus was isolated and deposited on June 14, 2006, at the Korean Agricultural Culture Collection (KACC) and the National Institute of Agricultural Biotechnology (NIAB) (Registration No.: KACC 97001P). Although this bacteriophage is effective in preventing and treating Staphylococcus aureus infections, its use has some limitations.

[0005] Antimicrobial proteins with killing activity against Staphylococcus aureus are derived from this bacteriophage, and these antimicrobial proteins can be used for the prevention and treatment of diseases caused by Staphylococcus aureus. See U.S. Patent No. 8,232,370.

[0006] In addition, this antimicrobial protein exhibits specific antimicrobial activity against all of the following genera: Staphylococcus aureus, Staphylococcus auriculi, Staphylococcus carinatum, Staphylococcus capsulatum, Staphylococcus chromogenicus, Staphylococcus cloacae, Staphylococcus dolphinus, Staphylococcus epidermidis, Staphylococcus equineus, Staphylococcus agalactiae, Staphylococcus hemolyticus, Staphylococcus hominis, Staphylococcus intermedia, Staphylococcus krusei, Staphylococcus stolonifera, Staphylococcus ludens, Staphylococcus flytraphytus, Staphylococcus pasteurellosis, saprophytic Staphylococcus, Staphylococcus var. ...

[0007] When preparing pharmaceutical compositions containing antimicrobial proteins, the composition must be formulated in a manner that maintains the activity of the antimicrobial protein for an appropriate period of time. Loss of activity or stability of the antimicrobial protein can result from the protein's chemical or physical instability, such as due to denaturation, aggregation, or oxidation. Therefore, the composition may be pharmaceutically unacceptable. The use of excipients is known to increase the stability of biologically active proteins; however, the stabilizing effect of these excipients is unpredictable and highly dependent on the properties of both the biologically active protein and the excipient.

[0008] There is still a need for formulations containing antimicrobial proteins as active ingredients, and for these formulations to be stable and suitable for injection over an appropriate period of time. The formulations can be used for administration to treat diseases caused by bacterial infections. Summary of the Invention

[0009] This invention provides a freeze-dried formulation comprising an antimicrobial protein having specific bactericidal activity against at least one or all of the following genera: *Staphylococcus aureus*, *Staphylococcus auriculata*, *Staphylococcus aureus*, *Staphylococcus carinatum*, *Staphylococcus capsulatum*, *Staphylococcus chromogenic*, *Staphylococcus cloacae*, *Staphylococcus dolphinii*, *Staphylococcus epidermidis*, *Staphylococcus equi*, *Staphylococcus gallinarum*, *Staphylococcus hemolyticus*, *Staphylococcus hominis*, *Staphylococcus intermedia*, *Staphylococcus krusei*, *Staphylococcus slow-acting*, *Staphylococcus ludensburgensis*, *Staphylococcus flytraides*, *Staphylococcus pasteurellosis*, *Staphylococcus saprophyticus*, *Staphylococcus var. ...

[0010] On the one hand, the concentration of antimicrobial protein in the solution prior to freeze-drying is approximately 0.1 mg / mL to approximately 30 mg / mL.

[0011] On the other hand, the antimicrobial protein consists of the amino acid sequence of SEQ ID NO:1.

[0012] On the other hand, the antimicrobial protein consists of the amino acid sequence of SEQ ID NO:2.

[0013] On the other hand, the antimicrobial protein is a mixture of a first antimicrobial protein consisting of the amino acid sequence of SEQ ID NO:1 and a second antimicrobial protein consisting of the amino acid sequence of SEQ ID NO:2.

[0014] On the other hand, the antimicrobial protein includes 15-35 mol% of a primary antimicrobial protein and 65-85 mol% of a secondary antimicrobial protein.

[0015] On the other hand, the antimicrobial protein includes 25 mol% of a primary antimicrobial protein and 75 mol% of a secondary antimicrobial protein.

[0016] On the other hand, the concentration of poloxamer in the solution prior to freeze-drying was approximately 0.1 g / L to approximately 10 g / L.

[0017] On the other hand, polosham is polosham 188.

[0018] On the other hand, the sugar is D-sorbitol.

[0019] On the other hand, the sugar concentration in the solution prior to freeze-drying is approximately 1 g / L to approximately 600 g / L.

[0020] On the other hand, the amino acid is L-histidine.

[0021] On the other hand, the concentration of amino acids in the solution prior to freeze-drying is approximately 0.1 g / L to approximately 10 g / L.

[0022] This invention provides an antimicrobial preparation comprising an antimicrobial protein having specific killing activity against at least one or all of the following genera: *Staphylococcus aureus*, *Staphylococcus auriculata*, *Staphylococcus aureus*, *Staphylococcus carinatum*, *Staphylococcus capsulatum*, *Staphylococcus chromogenic*, *Staphylococcus cloacae*, *Staphylococcus dolphus*, *Staphylococcus epidermidis*, *Staphylococcus equi*, *Staphylococcus gallinarum*, *Staphylococcus hemolyticus*, *Staphylococcus hominis*, *Staphylococcus intermedius*, *Staphylococcus krusei*, *Staphylococcus slow-acting*, *Staphylococcus ludun*, *Staphylococcus flytraphyta*, *Staphylococcus pasteurellosis*, *Staphylococcus saprophyticus*, *Staphylococcus var. var. var. xylose*; poloxamer; sugar; amino acids; and water. The antimicrobial protein consists of the amino acid sequence of SEQ ID NO:1, and the concentration of the antimicrobial protein is from about 0.1 mg / mL to about 30 mg / mL.

[0023] This invention provides an antimicrobial preparation comprising an antimicrobial protein having specific killing activity against at least one or all of the following genera: *Staphylococcus aureus*, *Staphylococcus auriculata*, *Staphylococcus aureus*, *Staphylococcus carinatum*, *Staphylococcus capsulatum*, *Staphylococcus chromogenic*, *Staphylococcus cloacae*, *Staphylococcus dolphus*, *Staphylococcus epidermidis*, *Staphylococcus equi*, *Staphylococcus gallinarum*, *Staphylococcus hemolyticus*, *Staphylococcus hominis*, *Staphylococcus intermedius*, *Staphylococcus krusei*, *Staphylococcus slow-acting*, *Staphylococcus ludun*, *Staphylococcus flytraphyta*, *Staphylococcus pasteurellosis*, *Staphylococcus saprophyticus*, *Staphylococcus var. var. var. xylose*; poloxamer; sugar; amino acids; and water. The antimicrobial protein consists of the amino acid sequence of SEQ ID NO:2, and the concentration of the antimicrobial protein is from about 0.1 mg / mL to about 30 mg / mL.

[0024] This invention provides an antimicrobial preparation comprising an antimicrobial protein having specific killing activity against at least one or all of the following genera: *Staphylococcus aureus*, *Staphylococcus auriculata*, *Staphylococcus aureus*, *Staphylococcus carinatum*, *Staphylococcus capsulatum*, *Staphylococcus chromogenic*, *Staphylococcus coli*, *Staphylococcus dolphus*, *Staphylococcus epidermidis*, *Staphylococcus equi*, *Staphylococcus gallinarum*, *Staphylococcus hemolyticus*, *Staphylococcus hominis*, *Staphylococcus intermedius*, *Staphylococcus krusei*, *Staphylococcus slow-acting*, *Staphylococcus ludun*, *Staphylococcus flytraphyta*, *Staphylococcus pasteurellium*, *Staphylococcus saprophyticus*, *Staphylococcus var. var. var. xylose*; poloxamer; sugar; amino acids; and water. The antimicrobial protein comprises a first antimicrobial protein consisting of the amino acid sequence of SEQ ID NO:1 and a second antimicrobial protein consisting of the amino acid sequence of SEQ ID NO:2, and the concentration of the antimicrobial protein is from about 0.1 mg / mL to about 30 mg / mL.

[0025] On one hand, the antimicrobial protein includes 15-35 mol% of a primary antimicrobial protein and 65-85 mol% of a secondary antimicrobial protein.

[0026] On the other hand, the antimicrobial protein includes 25 mol% of a primary antimicrobial protein and 75 mol% of a secondary antimicrobial protein.

[0027] On the other hand, polosham is polosham 188.

[0028] On the other hand, the concentration of poloxamer is from about 0.1 g / L to about 10 g / L.

[0029] On the other hand, the sugar is D-sorbitol.

[0030] On the other hand, the sugar concentration is from about 1 g / L to about 600 g / L.

[0031] On the other hand, the amino acid is L-histidine.

[0032] On the other hand, the concentration of amino acids is from about 0.1 g / L to about 10 g / L.

[0033] This application provides a method for manufacturing a freeze-dried formulation, comprising forming a mixture of: an antimicrobial protein having specific bactericidal activity against at least one or all of the following genera: *Staphylococcus aureus*, *Staphylococcus aureus*, *Staphylococcus auriculata*, *Staphylococcus carinatum*, *Staphylococcus capsulatum*, *Staphylococcus chromogenicus*, *Staphylococcus cloacae*, *Staphylococcus dolphinii*, *Staphylococcus epidermidis*, *Staphylococcus equi*, *Staphylococcus gallinarum*, *Staphylococcus hemolyticus*, *Staphylococcus hominis*, *Staphylococcus intermedia*, *Staphylococcus krusei*, *Staphylococcus slow-acting*, *Staphylococcus ludensburgensis*, *Staphylococcus flytraphytus*, *Staphylococcus pasteurellosis*, *Staphylococcus saprophyticus*, *Staphylococcus var. var. var. xylose*; poloxamer; sugar; and amino acids; and subjecting said mixture to freeze-drying.

[0034] On the one hand, the concentration of antimicrobial protein in the mixture prior to freeze-drying is from about 0.1 mg / mL to about 30 mg / mL.

[0035] On the other hand, the antimicrobial protein consists of the amino acid sequence of SEQ ID NO:1.

[0036] On the other hand, the antimicrobial protein consists of the amino acid sequence of SEQ ID NO:2.

[0037] On the other hand, the antimicrobial protein is a mixture of a first antimicrobial protein consisting of the amino acid sequence of SEQ ID NO:1 and a second antimicrobial protein consisting of the amino acid sequence of SEQ ID NO:2.

[0038] On the other hand, the antimicrobial protein includes 15-35 mol% of a primary antimicrobial protein and 65-85 mol% of a secondary antimicrobial protein.

[0039] On the other hand, the antimicrobial protein includes 25 mol% of a primary antimicrobial protein and 75 mol% of a secondary antimicrobial protein.

[0040] On the other hand, the concentration of poloxamer in the mixture prior to freeze-drying is from about 0.1 g / L to about 10 g / L.

[0041] On the other hand, polosham is polosham 188.

[0042] On the other hand, the sugar is D-sorbitol.

[0043] On the other hand, the sugar concentration in the mixture prior to freeze-drying is from about 1 g / L to about 600 g / L.

[0044] On the other hand, the amino acid is L-histidine.

[0045] On the other hand, the concentration of amino acids in the mixture prior to freeze-drying is from about 0.1 g / L to about 10 g / L.

[0046] It should be understood that both the foregoing general description and the following detailed description are illustrative and explanatory and are intended to provide further explanation of the claimed inventive technology. Attached Figure Description

[0047] The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and form part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[0048] In the diagram:

[0049] Figure 1 The results are from high-performance liquid chromatography (HPLC) analysis of the size exclusion of freeze-dried formulations at zero time.

[0050] Figure 2 The results were obtained by high-performance liquid chromatography (HPLC) analysis of the size exclusion of the freeze-dried formulation after one month of storage.

[0051] Figure 3 The results were obtained from high-performance liquid chromatography (HPLC) analysis of the size exclusion of the freeze-dried formulation after 6 months of storage. Detailed Implementation

[0052] Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings.

[0053] As used herein, “at least one or all of the following genera of Staphylococcus” means any one, two, three, four, five, six or up to twenty-two species of Staphylococcus selected from the following groups: Staphylococcus aureus, Staphylococcus auriculata, Staphylococcus carinatum, Staphylococcus capitis, Staphylococcus chromogenic, Staphylococcus cloacae, Staphylococcus dolphinus, Staphylococcus epidermidis, Staphylococcus equineus, Staphylococcus agalactiae, Staphylococcus hemolyticus, Staphylococcus hominis, Staphylococcus intermedia, Staphylococcus krillii, Staphylococcus stolonifera, Staphylococcus ludensundii, Staphylococcus flytraphytus, Staphylococcus pasteurellii, saprophytic Staphylococcus, Staphylococcus var. var. var., and Staphylococcus xylose.

[0054] Proteins are known to be relatively unstable in an aqueous state and undergo chemical and physical degradation, resulting in a loss of biological activity during processing and storage. Freeze-drying (also known as lyophilization) is a method for preserving proteins for storage.

[0055] Freeze-dried formulations include antimicrobial proteins having specific bactericidal activity against at least one or all of the following genera: *Staphylococcus aureus*, *Staphylococcus auriculata*, *Staphylococcus carinatum*, *Staphylococcus capsulatum*, *Staphylococcus chromogenic*, *Staphylococcus cloacae*, *Staphylococcus dolphus*, *Staphylococcus epidermidis*, *Staphylococcus equi*, *Staphylococcus gallinarum*, *Staphylococcus hemolyticus*, *Staphylococcus hominis*, *Staphylococcus intermedia*, *Staphylococcus krusei*, *Staphylococcus slow-acting*, *Staphylococcus ludun*, *Staphylococcus flytraides*, *Staphylococcus pasteurellosis*, *Staphylococcus saprophyticus*, *Staphylococcus var. var.*, and *Staphylococcus xylose*; poloxamer; sugars; and amino acids.

[0056] A method for manufacturing a freeze-dried formulation includes forming a mixture comprising: an antimicrobial protein having specific bactericidal activity against at least one or all of the following genera: *Staphylococcus aureus*, *Staphylococcus aureus*, *Staphylococcus auriculata*, *Staphylococcus carinatum*, *Staphylococcus capsulatum*, *Staphylococcus chromogenicus*, *Staphylococcus cloacae*, *Staphylococcus dolphinii*, *Staphylococcus epidermidis*, *Staphylococcus equi*, *Staphylococcus gallinarum*, *Staphylococcus hemolyticus*, *Staphylococcus hominis*, *Staphylococcus intermedia*, *Staphylococcus krusei*, *Staphylococcus stolonifera*, *Staphylococcus ludens*, *Staphylococcus flytraides*, *Staphylococcus pasteurellium*, *Staphylococcus saprophyticus*, *Staphylococcus var. var. var. xylose*; poloxamer; sugar; and amino acids; and subjecting said mixture to freeze-drying.

[0057] The concentration of antimicrobial protein in the solution prior to freeze-drying can be approximately 0.1 mg / mL to approximately 30 mg / mL, 0.1 mg / mL to 30 mg / mL, 0.5 mg / mL to 30 mg / mL, 1.0 mg / mL to 30 mg / mL, 1.5 mg / mL to 30 mg / mL, 5 mg / mL to 30 mg / mL, 0.1 mg / mL to 25 mg / mL, 0.1 mg / mL to 20 mg / mL, 0.5 mg / mL to 25 mg / mL, 0.5 mg / mL to 20 mg / mL, or 1.0 mg / mL to 20 mg / mL.

[0058] The antimicrobial protein consists of the amino acid sequence of SEQ ID NO:1, the amino acid sequence of SEQ ID NO:2, or a mixture of a first antimicrobial protein consisting of the amino acid sequence of SEQ ID NO:1 and a second antimicrobial protein consisting of the amino acid sequence of SEQ ID NO:2.

[0059] When the antimicrobial protein is a mixture of a first antimicrobial protein and a second antimicrobial protein, the antimicrobial protein may comprise 15-35 mol% of the first antimicrobial protein and 65-85 mol% of the second antimicrobial protein. For example, the antimicrobial protein may comprise 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 mol% of the first antimicrobial protein and 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85 mol% of the second antimicrobial protein.

[0060] Poloxamer is a monomeric triblock copolymer composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) and two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)). The concentration of poloxamer in the solution prior to freeze-drying can be from about 0.1 g / L to about 10 g / L, 0.1 g / L to 10 g / L, 0.2 g / L to 10 g / L, 0.1 g / L to 8 g / L, 0.2 g / L to 8 g / L, 0.1 g / L to 6 g / L, or 0.2 g / L to 6 g / L. Preferably, the poloxamer is poloxamer 188.

[0061] Preferred sugars used in freeze-dried formulations are, for example, D-sorbitol, sucrose, glucose, lactose, trehalose, glycerol, ethylene glycol, mannitol, xylitol, and inositol. More preferably, the sugar is D-sorbitol. The sugar concentration in the solution prior to freeze-drying can be from about 1 g / L to about 600 g / L, 1 g / L to 600 g / L, 5 g / L to 600 g / L, 1 g / L to 500 g / L, 5 g / L to 500 g / L, 1 g / L to 400 g / L, or 5 g / L to 400 g / L.

[0062] Preferred amino acids used in the freeze-dried formulation are, for example, L-histidine, L-glycine, and L-arginine. More preferably, the amino acid is L-histidine. The concentration of the amino acid in the solution prior to freeze-drying can be from about 0.1 g / L to about 10 g / L, 0.1 g / L to 10 g / L, 0.5 g / L to 10 g / L, 0.1 g / L to 8 g / L, 0.5 g / L to 8 g / L, 0.1 g / L to 6 g / L, or 0.5 g / L to 6 g / L.

[0063] Antimicrobial agents comprise antimicrobial proteins having specific bactericidal activity against at least one or all of the following genera: *Staphylococcus aureus*, *Staphylococcus auriculata*, *Staphylococcus aureus*, *Staphylococcus carinatum*, *Staphylococcus capsulatum*, *Staphylococcus chromogenic*, *Staphylococcus cloacae*, *Staphylococcus dolphus*, *Staphylococcus epidermidis*, *Staphylococcus equi*, *Staphylococcus gallinarum*, *Staphylococcus haemolyticus*, *Staphylococcus hominis*, *Staphylococcus intermedius*, *Staphylococcus krusei*, *Staphylococcus slow-acting*, *Staphylococcus ludun*, *Staphylococcus flytraides*, *Staphylococcus pasteurellosis*, *Staphylococcus saprophyticus*, *Staphylococcus var. var. var. xylose*; poloxamer; sugar; amino acids; and water. The antimicrobial protein consists of the amino acid sequence of SEQ ID NO:1, the amino acid sequence of SEQ ID NO:2, or comprises a first antimicrobial protein consisting of the amino acid sequence of SEQ ID NO:1 and a second antimicrobial protein consisting of the amino acid sequence of SEQ ID NO:2.

[0064] The concentration of antimicrobial protein in antimicrobial agents can be from about 0.1 mg / mL to about 30 mg / mL, 0.1 mg / mL to 30 mg / mL, 0.5 mg / mL to 30 mg / mL, 1.0 mg / mL to 30 mg / mL, 1.5 mg / mL to 30 mg / mL, 5 mg / mL to 30 mg / mL, 0.1 mg / mL to 25 mg / mL, 0.1 mg / mL to 20 mg / mL, 0.5 mg / mL to 25 mg / mL, 0.5 mg / mL to 20 mg / mL, or 1.0 mg / mL to 20 mg / mL.

[0065] When the antimicrobial protein is a mixture of a first antimicrobial protein and a second antimicrobial protein, the antimicrobial protein may comprise 15-35 mol% of the first antimicrobial protein and 65-85 mol% of the second antimicrobial protein. For example, the antimicrobial protein may comprise 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 mol% of the first antimicrobial protein and 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85 mol% of the second antimicrobial protein.

[0066] The concentration of poloxamer in the antibacterial preparation can be from about 0.1 g / L to about 10 g / L, 0.1 g / L to 10 g / L, 0.2 g / L to 10 g / L, 0.1 g / L to 8 g / L, 0.2 g / L to 8 g / L, 0.1 g / L to 6 g / L, or 0.2 g / L to 6 g / L. Preferably, the poloxamer is poloxamer 188.

[0067] Preferred sugars used in antimicrobial preparations are, for example, D-sorbitol, sucrose, glucose, lactose, trehalose, glycerol, ethylene glycol, mannitol, xylitol, and inositol. The concentration of sugars in antimicrobial preparations can be from about 1 g / L to about 600 g / L, 1 g / L to 600 g / L, 5 g / L to 600 g / L, 1 g / L to 500 g / L, 5 g / L to 500 g / L, 1 g / L to 400 g / L, or 5 g / L to 400 g / L.

[0068] Preferred amino acids used in antimicrobial preparations are, for example, L-histidine, L-glycine, and L-arginine. More preferably, the amino acid is L-histidine. The concentration of the amino acid in the antimicrobial preparation can be from about 0.1 g / L to about 10 g / L, 0.1 g / L to 10 g / L, 0.5 g / L to 10 g / L, 0.1 g / L to 8 g / L, 0.5 g / L to 8 g / L, 0.1 g / L to 6 g / L, or 0.5 g / L to 6 g / L.

[0069] A method for manufacturing a freeze-dried formulation includes forming a mixture comprising: an antimicrobial protein having specific bactericidal activity against at least one or all of the following genera: *Staphylococcus aureus*, *Staphylococcus aureus*, *Staphylococcus auriculata*, *Staphylococcus carinatum*, *Staphylococcus capsulatum*, *Staphylococcus chromogenicus*, *Staphylococcus cloacae*, *Staphylococcus dolphinii*, *Staphylococcus epidermidis*, *Staphylococcus equi*, *Staphylococcus gallinarum*, *Staphylococcus hemolyticus*, *Staphylococcus hominis*, *Staphylococcus intermedia*, *Staphylococcus krusei*, *Staphylococcus stolonifera*, *Staphylococcus ludens*, *Staphylococcus flytraides*, *Staphylococcus pasteurellium*, *Staphylococcus saprophyticus*, *Staphylococcus var. var. var. xylose*; poloxamer; sugar; and amino acids; and subjecting said mixture to freeze-drying.

[0070] The practical and currently preferred embodiments of the present invention are illustrated in the following examples.

[0071] However, it should be understood that, in light of this disclosure, modifications and improvements can be made by those skilled in the art within the spirit and scope of this invention.

[0072] Example 1: Preparation of antimicrobial protein

[0073] The expression plasmid for the antimicrobial protein of the present invention was constructed by conventionally cloning the gene encoding the antimicrobial protein of the present invention, shown in SEQ ID NO:3, into the pBAD-TOPO vector (Invitrogen). Escherichia coli BL21 cells transformed using the obtained plasmid were used as the host for the production of the antimicrobial protein of the present invention.

[0074] Using 0.2% arabinose at an optical density (OD) of 2.0 at 600 nm 600 The expression of the antimicrobial protein of the present invention was induced, and the induced bacterial cells were then incubated at 19°C for 10 hours. Bacterial cells were recovered by centrifugation (6,000×g, 20 min), and the resulting cell clumps were resuspended in a solubilization buffer [50 mM Na₂HPO₄ (pH 7.5), 10 mM EDTA, 1 mM dithiothreitol (DTT)] and disrupted by conventional sonication for 5 min (1-second pulse, 3-second rest interval between pulses). After centrifugation (13,000×g, 20 min), the supernatant was recovered and subjected to a two-step chromatography process including ion exchange chromatography (SP fast flow column; GE Healthcare) and hydrophobic interaction chromatography (Toyopearl PPG-600M column; Tosoh Bioscience).

[0075] For a more descriptive approach, the prepared host cells were inoculated into TSB (trypsin-soybean broth) medium (casein digest, 17 g / L; soybean digest, 3 g / L; dextrose, 2.5 g / L; NaCl, 5 g / L; potassium diphosphate, 2.5 g / L) and incubated at 37°C. The cells were cultured until the OD500 reached 2.0. 600At this point, L-arabinose was added to a final concentration of 0.2% to induce the expression of antimicrobial proteins. For induction, cells were cultured at 19°C for another 10 hours. The culture broth was centrifuged at 6,000×g for 20 minutes to obtain a cell pellet. The pellet was resuspended in 50mM Na₂HPO₄ buffer (pH 7.5) containing 10mM EDTA and 1mM DTT (10mL buffer per 1g of cells). Cells in the suspension were lysed using standard sonication. The cell lysate was centrifuged at 13,000×g for 20 minutes to remove cell debris. The supernatant was subjected to a two-step chromatographic process including: ion exchange chromatography (buffer A: 25 mM Na₂HPO₄ (pH 7.5), 10 mM EDTA; buffer B: 25 mM Na₂HPO₄ (pH 7.5), 10 mM EDTA, 1 M NaCl; buffer C: 25 mM Na₂HPO₄ (pH 7.5), 10 mM EDTA, 50 mM NaCl, 0.5% Triton X-100; program: sample loading → 1.6 CV of buffer A → 30 CV of buffer C → 20 CV of buffer A → 5 CV of 22% buffer B → gradient elution (20 CV of 22-100% buffer B)) and hydrophobic interaction chromatography (buffer A: 10 mM L-histidine (pH 7.5), 1 M NaCl; buffer B: 10 mM L-histidine (pH 7.5), 1 M NaCl, ... 7.5), 1M urea; Procedure: Sample loading (sample purification via ion exchange chromatography) → 10CV buffer A → gradient elution (10CV 0-100% buffer B). Then filter the protein solution using a 0.2μm filter.

[0076] To determine the composition of the antimicrobial protein consisting of the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:2, a two-step analysis was performed. First, the protein sample treated with a protease was subjected to liquid chromatography-mass spectrometry (LC-MS). The protein solution obtained according to the above procedure was centrifuged and filtered into 50 mM Tris-HCl buffer (pH 7.6) for buffer exchange and diluted to a concentration of 2.5 mg / mL with 6 M urea solution. The diluted protein solution was then treated with a protease. Sequencing-grade modified porcine Glu-C protease (Promega, Madison, WI, USA) was used as the protease, and protease treatment was performed according to the manufacturer's instructions. After protease treatment, the resulting protease-treated protein solution was subjected to reversed-phase HPLC and Q-TOF-MS. By peak analysis, the HPLC and MS peaks corresponding to the MAKTQAE peptide fragment of the antimicrobial protein consisting of the amino acid sequence of SEQ ID NO:1 and the AKTQAE peptide fragment of the antimicrobial protein consisting of the amino acid sequence of SEQ ID NO:2 were identified based on the estimated protease digestion pattern and mass. Furthermore, HPLC and MS peaks were confirmed by comparing peak chromatograms obtained using chemically synthesized peptides (MAKTQAE and AKTQAE) as samples. Subsequently, reverse-phase HPLC analysis of the protein samples treated with protease and the chemically synthesized peptides (MAKTQAE and AKTQAE) was performed to determine the composition ratio of the antimicrobial protein formulation consisting of the amino acid sequence of SEQ ID NO:1 based on the correlation between peptide concentration and the corresponding peak area. As a result of the analysis using three batches of antimicrobial protein, the composition ratio of the antimicrobial protein consisting of the amino acid sequence of SEQ ID NO:1 was determined to be 25, 27, and 29 mol%.

[0077] Example 2: Preparation of a pharmaceutical composition having a freeze-dried formulation

[0078] A pharmaceutical composition for treating staphylococcal infections, comprising the antimicrobial protein of the present invention, is prepared by freeze-drying. A freeze-dried formulation having the following composition is prepared:

[0079] Table 1

[0080]

[0081] The manufacturing process includes exchanging the protein solution buffer prepared in Example 1 with a buffer containing the aforementioned components, concentrating the obtained solution, adjusting the concentration of the antimicrobial protein in the solution, filtering the concentration-adjusted solution, and lyophilizing the filtrate.

[0082] The following is a description of each step of the process:

[0083] The protein solution buffer prepared in Example 1 was replaced with a buffer (1.56 g / L L-histidine (pH 6.0), 50 g / L D-sorbitol, 1.47 g / L CaCl2·2H2O and 1 g / L poloxamer 188) using conventional percolation.

[0084] The obtained solution was concentrated using a centrifugal filter (10K).

[0085] The concentration of the antimicrobial protein was adjusted to 18 mg / mL using a protein buffer (1.56 g / L L-histidine (pH 6.0), 50 g / L D-sorbitol, 1.47 g / L CaCl2·2H2O and 1 g / L poloxamer 188) based on the protein assay using conventional dicinchin (BCA).

[0086] The concentration-adjusted solution was filtered using a 0.2-μm filter.

[0087] Add 1 mL of filtered solution to a 3-mL glass vial and place the filled vial into a stainless steel tray.

[0088] Load the trays into the freeze dryer and use the following freeze-drying cycle to freeze-dry the product:

[0089] Equilibrate at 4°C for approximately 20 minutes.

[0090] Maintain the rack temperature at -40°C for 12 hours.

[0091] Set the condenser temperature to -50℃.

[0092] Apply a vacuum to the room.

[0093] When the vacuum reaches a value of 1,500 mtorr, the rack temperature is raised to -20°C and maintained for 16 hours.

[0094] The shelf temperature was increased to 20°C at a rate of 10°C per hour and maintained for 4 hours.

[0095] Break the vacuum.

[0096] Use the appropriate flip-top cap to plug and seal the vial with the stopper.

[0097] The lyophilized formulation was stored at 4°C, and its stability and bioactivity were tested as indicated below. The composition was reconstituted using water for injection (0.92 mL) prior to analysis. Stability was determined using size exclusion high-performance liquid chromatography (SEC-HPLC). BioSep was used for further analysis. TMSEC-HPLC was performed on a SEC-S 2000 column (Phenomenex, Torrance, CA). The mobile phase (10 mM Tris, 0.5 M NaCl, 1 M urea, pH 7.5) was applied at a flow rate of 1.0 mL / min. 50 μL of sample was injected, and elution was monitored by measuring absorbance at 280 nm for 30 min. Results are shown in [Table data would be inserted here]. Figure 1-3 middle.

[0098] Biological activity was analyzed using a turbidity reduction assay. The turbidity reduction assay was performed as follows: The sample was added to 10 mM phosphate-buffered saline (PBS) (pH 7.2) to Staphylococcus aureus strain ATCC 33591 (OD2000). 600 In a suspension containing 0.5 g of antimicrobial protein, the final concentration was 0.1 μg / mL. Bacterial cell density (OD) was recorded every 30 seconds. 600 The changes in (TOD) were recorded over a total of 15 minutes. TOD was obtained from this experiment. 50 (Half logarithmic decrease in the initial concentration of live bacteria, expressed in minutes).

[0099] Table 2 summarizes the results of analytical tests related to the stability and bioactivity of the formulation. Values ​​were determined at four checkpoints: at zero, after 1 month, 3 months, and 6 months of storage at 4°C. In the stability test, the intact protein content at zero was considered 100%. In the bioactivity test, the TOD (Total Organic Protein) measured at zero was analyzed. 50 Differences in values.

[0100] Table 2

[0101] Stability and bioactivity

[0102]

[0103] As can be inferred from Table 2, the stability and bioactivity of the freeze-dried formulation of the present invention are well preserved after 6 months of storage.

[0104] Example 3: Comparison of freeze-dried formulations and liquid formulations

[0105] The bioactivity of the lyophilized formulation and the liquid formulation was compared using the turbidity reduction assay described in Example 2. The lyophilized formulation was used after being stored for one month. It was reconstituted with water for injection (0.92 mL) before bioactivity analysis. The liquid formulation was prepared freshly using filtered solution according to the procedure described in Example 2. The following bacterial strains were used in this experiment.

[0106] Table 3

[0107] Test strain

[0108]

[0109]

[0110] In the turbidity reduction assay, the final antimicrobial protein concentration applied was 0.1 μg / mL for the following strains: Staphylococcus aureus, Staphylococcus auriculata, Staphylococcus carinatum, Staphylococcus capsulatum, Staphylococcus chromogenic, Staphylococcus dolphinus, Staphylococcus epidermidis, Staphylococcus equi, Staphylococcus gallinarum, Staphylococcus hemolyticus, Staphylococcus hominis, Staphylococcus krusei, Staphylococcus ludensii, Staphylococcus viridis, Staphylococcus saprophyticus, and Staphylococcus xylose. For tests against Staphylococcus aureus, Staphylococcus coli, Staphylococcus intermedius, Staphylococcus stenoticus, and Staphylococcus var. ... 50 Value differences. The results are provided in Table 4.

[0111] Table 4

[0112]

[0113]

[0114] The results shown in Table 4 clearly indicate that the freeze-dried formulations of the present invention can provide antibacterial activity and efficacy very similar to those of the liquid formulations. Furthermore, the results shown in Table 4 demonstrate that the freeze-dried formulations of the present invention exhibit rapid and effective bactericidal activity against various Staphylococcus strains. The TOD50 of the freeze-dried formulations of the present invention against the tested Staphylococcus strains is approximately no more than 20 minutes.

[0115] Simultaneously, the antibacterial activity of the freeze-dried formulation of the present invention against non-staphylococcal strains was examined. Two *Enterococcus faecalis* strains, three *Enterococcus faecium* strains, two *Streptococcus viridans* strains, one *Streptococcus lactis* strain, five *Escherichia coli* strains, two *Clostridium perfringens* strains, and three *Salmonella* strains were tested as non-staphylococcal strains. Therefore, the freeze-dried formulation of the present invention showed no antibacterial activity against these tested non-staphylococcal strains (Table 5). This result indicates that the antibacterial activity of the freeze-dried formulation of the present invention is specific to *Staphylococcus*.

[0116] Table 5

[0117] Antimicrobial activity against non-staphylococcal strains

[0118]

[0119] Therefore, it is inferred that the freeze-dried formulation of the present invention is specific to Staphylococcus and has a broad antibacterial spectrum within Staphylococcus, indicating that the freeze-dried formulation of the present invention can be used as a therapeutic agent for Staphylococcus infections.

[0120] Example 4: Therapeutic effect of freeze-dried formulation on single staphylococcal infections

[0121] The therapeutic effect of the freeze-dried formulation of the present invention on single staphylococcal infections was studied using animal models. In this experiment, *Staphylococcus epidermidis* and *Staphylococcus hemolyticus* were selected as model staphylococcal strains. The freeze-dried formulation was used after being stored for one month. It was reconstituted with water for injection (0.92 mL) before use in animal experiments. A freshly prepared filtered solution according to the procedure described in Example 2 was used as the liquid formulation.

[0122] For the Staphylococcus epidermidis assay, female ICR mice weighing 23 g ± 20% (5 weeks old) [SPF-free pathogen grade] were used. A total of 30 mice (10 mice per group) were intravenously injected with an inoculum of Staphylococcus epidermidis strain CCARM 3751 (1 × 10⁻⁶). 8 CFU / mouse). At 30 minutes, 12 hours, and 24 hours post-challenge, a control group of animals was administered buffer-only (1.56 g / L L-histidine (pH 6.0), 50 g / L D-sorbitol, 1.47 g / L CaCl2·2H2O, and 1 g / L poloxamer 188) intravenously three times. At 30 minutes, 12 hours, and 24 hours post-challenge, a treatment group using the reconstituted solution of the lyophilized formulation was administered intravenously three times (dose: 25 mg / kg). At 30 minutes, 12 hours, and 24 hours post-challenge, a treatment group using the liquid formulation was administered intravenously three times (dose: 25 mg / kg). The number of dead mice was recorded, and clinical signs were observed daily. The ability of the reconstituted solution of the lyophilized formulation and the liquid formulation to eradicate bacteria from the bloodstream was assessed using blood collected 5 days post-challenge (the experimental endpoint) via routine community counting.

[0123] For the Staphylococcus aureus assay, female ICR mice weighing 22 g ± 20% (5 weeks old) [SPF-free pathogen grade] were used. A total of 30 mice (10 mice per group) were intravenously injected with an inoculum of Staphylococcus aureus strain CCARM 3733 (1 × 10⁻⁶). 8CFU / mouse). At 30 minutes, 12 hours, and 24 hours post-challenge, a control group of animals was administered buffer-only (1.56 g / L L-histidine (pH 6.0), 50 g / L D-sorbitol, 1.47 g / L CaCl2·2H2O, and 1 g / L poloxamer 188) intravenously three times. At 30 minutes, 12 hours, and 24 hours post-challenge, a treatment group using the reconstituted solution of the lyophilized formulation was administered intravenously three times (dose: 25 mg / kg). At 30 minutes, 12 hours, and 24 hours post-challenge, a treatment group using the liquid formulation was administered intravenously three times (dose: 25 mg / kg). The number of dead mice was recorded, and clinical signs were observed daily. The ability of the reconstituted solution of the lyophilized formulation and the liquid formulation to eradicate bacteria from the bloodstream was assessed using blood collected 5 days post-challenge (the experimental endpoint) via routine community counting.

[0124] As a result, a significant therapeutic effect was observed. The two experiments showed similar results. Regarding clinical signs, although mice in the treatment group remained normal throughout the experimental period, mice in the control group began to exhibit various clinical signs 2 days after bacterial challenge, including eyelid erythema, reduced spontaneous activity, hair loss, piloerection, and circling. Intravenous injection of the reconstituted solution of the lyophilized formulation and the liquid formulation significantly improved survival rates (Table 6).

[0125] Table 6

[0126] Mortality rate in a single Staphylococcus infection model experiment

[0127]

[0128] Furthermore, intravenous injection of the reconstituted solution of the freeze-dried formulation and the liquid formulation significantly reduced bacterial counts in the blood. The mean CFU / mL in serum collected from control mice in the Staphylococcus epidermidis experiment was >1×10⁻⁶. 6 Furthermore, the serum from control mice in the Staphylococcus aureus experiment was >1×10⁻⁶. 5 In contrast, no bacterial community was observed in mice in any of the treatment groups.

[0129] The above results confirm that the freeze-dried formulation of the present invention provides a therapeutic effect very similar to that of the liquid formulation in treating single staphylococcal infections. Furthermore, the results shown in Table 6 demonstrate that the freeze-dried formulation of the present invention can be effectively used to treat staphylococcal infections.

[0130] Example 5: Therapeutic effects of freeze-dried formulation on various Staphylococcus infections

[0131] The therapeutic effects of the freeze-dried formulation of the present invention on various staphylococcal infections were studied using animal models. In this experiment, *Staphylococcus epidermidis*, *Staphylococcus ludens*, and *Staphylococcus war netilé* were selected as model staphylococcal strains. The freeze-dried formulation was used after being stored for one month. It was reconstituted with water for injection (0.92 mL) before use in animal experiments. A freshly prepared filtered solution according to the procedure described in Example 2 was used as the liquid formulation.

[0132] Female ICR mice weighing 22g ± 20% (5 weeks old) [SPF grade] were used. A total of 30 mice (n=10 per group) were intravenously injected with a mixed inoculum of Staphylococcus epidermidis CCARM 3751, Staphylococcus ludensii CCARM 3734, and Staphylococcus warwickii KCTC 3340 (ATCC 27836) (1×10⁻⁶ each). 8 CFU / mouse). At 30 minutes, 12 hours, and 24 hours post-challenge, a control group of animals was administered buffer-only (1.56 g / L L-histidine (pH 6.0), 50 g / L D-sorbitol, 1.47 g / L CaCl2·2H2O, and 1 g / L poloxamer 188) intravenously three times. At 30 minutes, 12 hours, and 24 hours post-challenge, a treatment group using the reconstituted solution of the lyophilized formulation was administered intravenously three times (dose: 25 mg / kg). At 30 minutes, 12 hours, and 24 hours post-challenge, a treatment group using the liquid formulation was administered intravenously three times (dose: 25 mg / kg). The number of dead mice was recorded, and clinical signs were observed daily. The ability of the reconstituted solution of the lyophilized formulation and the liquid formulation to eradicate bacteria from the bloodstream was assessed using blood collected 5 days post-challenge (the experimental endpoint) via routine community counting.

[0133] As a result, a significant therapeutic effect was observed. Regarding clinical signs, while mice in the treatment group remained normal throughout the experimental period, mice in the control group exhibited various clinical signs, including eyelid erythema, reduced spontaneous activity, hair loss, ptosis, and piloerection. Intravenous injection of the reconstituted solution of the freeze-dried formulation and the liquid formulation significantly improved survival rates (Table 7).

[0134] Table 7

[0135] Mortality rates in various Staphylococcus infection model experiments

[0136]

[0137] Furthermore, intravenous injection of the reconstituted solution of the lyophilized formulation and the liquid formulation significantly reduced bacterial counts in the blood. The mean CFU / mL in serum collected from control mice was >1×10⁻⁶. 6 In contrast, no bacterial communities were observed in any of the treatment groups of mice.

[0138] The above results confirm that the freeze-dried formulation of the present invention provides a therapeutic effect very similar to that of the liquid formulation in treating various staphylococcal infections. Furthermore, the results shown in Table 7 demonstrate that the freeze-dried formulation of the present invention can be effectively used to treat staphylococcal infections.

[0139] Those skilled in the art will appreciate that various modifications and variations can be made to this invention without departing from its spirit or scope. Therefore, this invention is intended to cover modifications and variations thereof, provided they fall within the scope of the appended claims and their equivalents.

Claims

1. A method for manufacturing a freeze-dried formulation, comprising: An antimicrobial protein solution is prepared, wherein the antimicrobial protein solution is a mixture of a first antimicrobial protein and a second antimicrobial protein, the first antimicrobial protein being composed of the amino acid sequence of SEQ ID NO: 1, and the second antimicrobial protein being composed of the amino acid sequence of SEQ ID NO: 2, wherein the mixture of the first and second antimicrobial proteins has bactericidal activity against all of the following Staphylococcus spp.: *Staphylococcus aureus*, *Staphylococcus auriculata*, *Staphylococcus carinatum*, *Staphylococcus capsulatum*, *Staphylococcus chromogenic*, *Staphylococcus cloacae*, *Staphylococcus dolphinii*, *Staphylococcus epidermidis*, *Staphylococcus equi*, *Staphylococcus gallinarum*, *Staphylococcus hemolyticus*, *Staphylococcus hominis*, *Staphylococcus intermedius*, *Staphylococcus krusei*, *Staphylococcus slow-acting*, *Staphylococcus ludun*, *Staphylococcus flytraphyta*, *Staphylococcus pasteurellosis*, *Staphylococcus saprophyticus*, *Staphylococcus var. var. var.*, and *Staphylococcus xylose*; wherein the preparation of the mixture of the first and second antimicrobial proteins includes: An expression plasmid for the antimicrobial protein was constructed by routinely cloning the gene encoding the antimicrobial protein shown in SEQ ID NO:3 into the pBAD-TOPO vector. *E. coli* BL21 cells transformed using the obtained plasmid were used as the host cell for antimicrobial protein production. The prepared host cells were inoculated into TSB medium and incubated at 37°C. When the cell concentration reached an optical cell density of 2.0 g / L at 600 nm, L-arabinose was added to the medium at a final concentration of 0.2% to induce antimicrobial protein expression. For further induction, the cells were cultured at 19°C for another 10 hours. The TSB medium consisted of 17 g / L casein digest, 3 g / L soybean digest, 2.5 g / L dextrose, 5 g / L NaCl, and 2.5 g / L potassium diphosphate. Furthermore, the mixture of the first antimicrobial protein and the second antimicrobial protein consists of 25-29 mol% of the first antimicrobial protein and 71-75 mol% of the second antimicrobial protein; Preparation of a solution comprising poloxamer 188, D-sorbitol, L-histidine, and a mixture of the first antimicrobial protein and the second antimicrobial protein, wherein the preparation of the solution comprises: The antimicrobial protein solution buffer of the mixture of the first and second antimicrobial proteins was exchanged with a buffer containing 1 g / L poloxamer 188, 50 g / L D-sorbitol, 1.56 g / L L-histidine, and 1.47 g / L CaCl2·2H2O; the buffer-exchanged solution was concentrated using a centrifugal filter; the concentrations of the first and second antimicrobial proteins were adjusted to 18 mg / mL; the concentration-adjusted solution was filtered using a 0.2 μm filter; and The solution comprising 1 g / L poloxamer 188, 50 g / L D-sorbitol, 1.56 g / L L-histidine, 1.47 g / L CaCl2·2H2O, and a mixture of 25-29 mol% of the first antimicrobial protein and 71-75 mol% of the second antimicrobial protein was freeze-dried to obtain a freeze-dried formulation, wherein the freeze-dried formulation contained 99.8% of the intact protein and less than 5% reduction in biological activity after 6 months.

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