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  Technology for preparation of macromolecular microspheresUSPTO Application #: 20070190163Title: Technology for preparation of macromolecular microspheres Abstract: Microspheres are produced by contacting an aqueous solution of a protein or other macromolecule with an organic solvent and a counterion, and chilling the solution. The microspheres are useful for preparing pharmaceuticals of defined dimensions. (end of abstract)
Agent: Fish & Richardson, PC - Minneapolis, MN, US Inventors: Michael P. Malaknov, Fang Fang USPTO Applicaton #: 20070190163 - Class: 424499000 (USPTO) Related Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Preparations Characterized By Special Physical Form, Particulate Form (e.g., Powders, Granules, Beads, Microcapsules, And Pellets), Contains Proteins Or Derivative Or Polysaccharides Or Derivative The Patent Description & Claims data below is from USPTO Patent Application 20070190163. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATIONS [0001] Benefit of priority under 35 U.S.C. .sctn.119(e) is claimed to U.S. Provisional Application No. 60/762,002, filed 24 Jan. 2006, entitled "Technology for Preparation of Macromolecular Microspheres." This provisional application is incorporated by reference in its entirety. [0002] This application also is related to International PCT application Application Serial No. (Attorney Docket No. 21865-004WO1/6504PC, filed 24 Jan. 2007). This application also is related to published U.S. applications Serial Nos. US20050004020 A1 and US20050112751 A1. Each of these applications is incorporated by reference in its entirety. BACKGROUND [0003] The administration of proteins to animals, including humans, in nutritional supplements or as therapeutics has been known for some time. Proteins for therapeutic or nutritional administration generally are available either as (1) concentrates or powders that are administered directly or are reconstituted in a liquid of choice prior to use; or (2) liquid formulations. [0004] The preparation and delivery of therapeutic proteins of interest in powder or particle form is an area of concentrated research and development activity in the pharmaceutical industry. For therapeutic efficacy, it is desirable to have a uniform formulation. For example, for pulmonary administration, the protein ideally is prepared in the form of discrete microspheres, which are solid or semi-solid particles having a diameter of between 0.5 and 5.0 microns. It also is desirable for the particles to have a protein content that is as high as possible and that maintains its activity for concentrated delivery and therapeutic efficacy. [0005] Previous methods of producing protein microparticles or nanoparticles have involved complex steps, such as blending with organic polymers and/or forming a lattice array with polymers; spray drying, spray freeze-drying or supercritical fluid antisolvent techniques that use specialized and complex equipment; or lyophilization followed by pulverization or milling that often results in non-uniform particles that must further be sorted. Often previous methods of producing solid protein formulations involve processing steps, such as heating, that denature the protein and compromise its activity. In addition, some methods do not provide high recovery from solution into the solid formulation. [0006] Accordingly, there is a need for a method for producing protein and other macromolecular microparticles that does not require complex or specialized equipment and that produces uniform-sized microparticles for delivery. There further is a need for a method of producing microparticles that contain high concentrations of the protein or macromolecule relative to other components, that are stable and maintain their activity for long periods of time when stored at ambient temperature and that do not contain a significant amount of denatured protein. There also is a need for a method of producing microparticles of proteins and other macromolecules wherein substantially all of the protein or macromolecule in the starting material is recovered in the microparticle formulation, with minimal loss. There also is a need for microparticles of proteins or other macromolecules containing these properties for administration, for example, as a therapeutic or nutritional supplement. SUMMARY [0007] Provided herein are methods for producing protein and other macromolecular microparticles that do not require complex or specialized equipment and that produces uniform-sized microparticles for delivery; methods for producing microparticles that contain high concentrations of protein or macromolecule relative to other components, that are stable and maintain their activity for long periods of time when stored at ambient temperature and that do not contain a significant amount of denatured protein. Also provide are methods for producing microparticles of proteins and other macromolecules where substantially all of the protein or macromolecule in the starting material is recovered in the microparticle formulation, with minimal loss. Also provided are microparticles of proteins and other macromolecules containing these properties for administration, for example, as a therapeutic or nutritional supplement. [0008] The methods of making a protein-based composition, the protein-based compositions themselves, combinations, articles of manufacture provided below are characterized by a variety of component ingredients, steps of preparation, and biophysical, physical, biochemical and chemical parameters. As would be apparent to one of skill in the art, the compositions and methods provided herein include any and all permutations and combinations of the ingredients, steps and/or parameters described below. [0009] Provided herein are methods of making a protein-based composition. The method provided herein can be used to make compositions from other macromolecules besides proteins, including DNA, RNA, PNA, lipids, oligosaccharides and combinations thereof. [0010] The methods provided herein can include the steps of: [0011] a) adding a counterion to a solution containing the protein in an aqueous solvent; [0012] b) adding an organic solvent to the solution; and [0013] c) gradually cooling the solution to a temperature below about 25.degree. C., whereby a composition containing microparticles comprising the protein is formed, wherein steps a), b) and c) are performed simultaneously, sequentially, intermittently, or in any order. [0014] In one embodiment, the steps are performed sequentially a), b) and then c). In another embodiment, the method of making a protein-based composition includes performing steps a) and b) simultaneously or sequentially in any order, followed by step c). [0015] The resulting microparticles can be obtained by precipitation, by phase separation or by colloid formation. In some aspects, the methods provided herein further comprise separating the microparticles from the solution to remove components other than the microparticles. This separation step can be performed following the above-mentioned step c). The separation can be effected by, for example, sedimentation, filtration and/or freeze-drying. [0016] The methods provided herein include the addition of an organic solvent to an aqueous solvent containing the protein. In certain embodiments, the organic solvent in miscible or partially miscible with the aqueous solvent. In further embodiments of the methods provided herein, the organic solvent is selected from among aliphatic alcohols, aromatic alcohols, chloroform, dimethyl chloride, polyhydric sugar alcohols, aromatic hydrocarbons, aldehydes, ketones, esters, ethers, dioxanes, alkanes, alkenes, conjugated dienes, dichloromethane, acetonitrile, ethyl acetate, polyols, polyimides, polyesters, polyaldehydes and mixtures thereof. For example, where the organic solvent is an aliphatic alcohol, the organic solvent can be isopropanol. The amount of organic solvent added can vary in the methods provided herein. For example, the amount of organic solvent added can be from about 0.1% or 0.1% to about 50% or 50% v/v. In other embodiments, the amount of organic solvent added is from about 1% or 1% to about 30% or 30% v/v, from about 5% or 5% to about 30% or 30% v/v, from about 10% or 10% to about 30% or 30% v/v or from about 15% or 15% to about 20% or 20% v/v. [0017] The counterion used in the methods provided herein can be an anionic compound, a cationic compound and/or a zwitterionic compound. For example, when the counterion is an anionic compound, the counterion can be glycine, sodium citrate, sodium sulfate, zinc sulfate, magnesium sulfate, potassium sulfate or calcium sulfate. The concentration of organic solvent added to the solution can vary in the method provided herein. For example, the concentration of counterion added to the solution can be from about 0.1 mM or 0.1 mM to about 100 mM or 100 mM. In other embodiments, the concentration of counterion added to the solution is from about 0.2 mM or 0.2 mM to about 50 mM or 50 mM, from about 0.3 mM or 0.3 mM to about 30 mM or 30 mM, from about 0.5 mM or 0.5 mM to about 20 mM or 20 mM or from about 1 mM or 1 mM to about 10 mM or 10 mM. In a particular embodiment, the concentration of counterion added to the solution is about 5 mM or 5 mM. [0018] In one aspect of the methods provided herein, the pH of the solution that contains the protein is at or below the pI of the protein. In some aspects, the pH of the solution is from about 4.0 or 4.0 to about 9.0 or 9.0. In other aspects, the pH of the solution is from about 4.5 or 4.5 to about 8.0 or 8.0, from about 4.5 or 4.5 to about 6.5 or 6.5, or from about 4.5 or 4.5 to about 5.5 or 5.5. [0019] The protein that is used in the methods provided herein to make a protein-based composition can be selected from among sialidases, sialidase fusion proteins, proteases, protease inhibitors, cytokines, insulin, human growth hormone, calcitonin, recombinant human DNase, interferons and parathyroid hormone. In one embodiment, where the protein is a protease inhibitor, the protein is human protease inhibitor 8 (PI8). In another embodiment, the protein is a sialidase fusion protein. In some aspects where the protein is a sialidase fusion protein, the sialidase fusion protein contains a catalytic domain of a sialidase and an anchoring domain, wherein the catalytic domain of the sialidase is the only portion of the sialidase in the sialidase fusion protein. The sialidase can be, for example, an Actinomyces viscosus sialidase, a Clostridium perfringens sialidase, an Arthrobacter ureafaciens sialidase, a Micromonospora viridifaciens sialidase, a human Neu2 sialidase or a human Neu4 sialidase. [0020] In one aspect, where the sialidase is an Actinomyces viscosus sialidase, the amino acid sequence of the catalytic domain contains the sequence of amino acid residues beginning at any of the amino acids from amino acid 270 to amino acid 290 and ending at any of the amino acids from amino acid 665 to amino acid 901 of the sequence of amino acids set forth in SEQ ID NO. 1. For example, in one embodiment, the sequence of the sialidase catalytic domain contains the sequence of amino acid residues set forth in SEQ ID NO:2. In another embodiment, the sequence of the catalytic domain comprises the sequence of amino acid residues beginning at amino acid 274 and ending at amino acid 681 of the sequence of amino acids set forth in SEQ ID NO. 1. In a different embodiment, the sequence of the catalytic domain comprises the sequence of amino acid residues beginning at amino acid 274 and ending at amino acid 666 of the sequence of amino acids set forth in SEQ ID NO. 1. In another embodiment, the sequence of the catalytic domain comprises the sequence of amino acids beginning at amino acid 290 and ending at amino acid 681 of the sequence of amino acids set forth in SEQ ID NO. 1. [0021] In one aspect, where the protein that is used in the methods provided herein to make a protein-based composition is a sialidase fusion protein that contains an anchoring domain, the anchoring domain is a glycosaminoglycan (GAG)-binding domain. In a further aspect, the GAG-binding domain is selected from among the GAG-binding domain of human platelet factor 4, the GAG-binding domain of human interleukin 8, the GAG-binding domain of human antithrombin III, the GAG-binding domain of human apoprotein E, the GAG-binding domain of human angio-associated migratory protein and the GAG-binding domain of human amphiregulin. In particular embodiments, the amino acid sequence of the GAG-binding domain contains the sequence of amino acid residues set forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8. Continue reading... Full patent description for Technology for preparation of macromolecular microspheres Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Technology for preparation of macromolecular microspheres patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. 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