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.

1 . (canceled) 
     
     
         2 . A method of making protein-based microparticles, comprising:
 a) mixing together: (i) a solution containing a protein in an aqueous solvent, (ii) a counterion and (iii) one or more organic solvents, wherein each organic solvent in the resulting mixture is volatile and none are a polymer; and   b) providing the resulting mixture at or cooling the resulting mixture to a temperature below about 25° C., whereby microparticles comprising the protein are formed.   
     
     
         3 . The method of  claim 2 , wherein the organic solvent is selected from among methanol, ethanol, 1-propanol, isopropanol, tert-butyl alcohol, butanol, acetone, acetonitrile, acetic acid, ethyl acetate, methyl ethyl ketone, methyl t-butyl ether, and dimethyl sulfoxide. 
     
     
         4 . The method of  claim 2 , wherein the counterion is an anionic compound. 
     
     
         5 . The method of  claim 4 , wherein the counterion is selected from among citrate, phosphate, sulfate, pivalate, rubidium, bromine, perchlorate, triethylamine, glycine, itaconate, and arginine, and any salt, acid, or base form thereof. 
     
     
         6 . The method of  claim 2 , wherein the protein is a sialidase fusion protein. 
     
     
         7 . The method of  claim 6 , wherein the sequence of the sialidase fusion protein comprises the sequence of amino acids set forth in SEQ ID NO:17. 
     
     
         8 . The method of  claim 2 , wherein the temperature is between about 4° C. to about −45° C. 
     
     
         9 . The method of  claim 2 , further comprising adding an active agent to step a) or to a suspension of the formed microspheres, wherein the active agent is selected from among antidiabetics, anticonvulsants, analgesics, antiparkinsons, anti-inflammatories, calcium antagonists, anesthetics, antimicrobials, antimalarials, antiparasitics, antihypertensives, antihistamines, antipyretics, alpha-adrenergic agonists, alpha-blockers, biocides, bactericides, bronchial dilators, beta-adrenergic blocking drugs, contraceptives, cardiovascular drugs, calcium channel inhibitors, depressants, diuretics, electrolytes, enzymes, hormones, hypoglycemics, hyperglycemics, muscle contractants, muscle relaxants, antineoplastics, glycoproteins, nucleoproteins, lipoproteins, ophthalmics, sedatives, steroids, sympathomimetics, parasympathomimetics, tranquilizers, vaccines, vitamins, minerals, nonsteroidal anti-inflammatory drugs, angiotensin converting enzymes, polynucleotides, polypeptides, polysaccharides and nutritional supplements; whereby the microspheres comprise the active agent. 
     
     
         10 . The method of  claim 2 , wherein the organic solvent is present in the mixture at from about 0.1% to about 50% v/v. 
     
     
         11 . The method of  claim 10 , wherein the organic solvent is present in the mixture at about 1% to about 30% v/v. 
     
     
         12 . The method of  claim 2 , wherein the size of the microparticles is from about 0.5 μm to about 5.0 μm. 
     
     
         13 . A method of making protein-based microparticles, comprising:
 a) providing a solution comprising a protein, a counterion, an aqueous solvent and one or more organic solvents, wherein each organic solvent present in the solution is a volatile solvent other than ethanol and none are a polymer; and   b) providing the solution at or cooling the solution to a temperature below about 25° C., whereby microparticles comprising the protein are formed.   
     
     
         14 . The method of  claim 13 , wherein the organic solvent is selected from among methanol, 1-propanol, isopropanol, tert-butyl alcohol, butanol, acetone, acetonitrile, acetic acid, ethyl acetate, methyl ethyl ketone, methyl t-butyl ether, and dimethyl sulfoxide. 
     
     
         15 . The method of  claim 13 , wherein the counterion is an anionic compound. 
     
     
         16 . The method of  claim 15 , wherein the counterion is selected from among citrate, phosphate, sulfate, pivalate, rubidium, bromine, perchlorate, triethylamine, glycine, itaconate, and arginine, and any salt, acid, or base form thereof. 
     
     
         17 . The method of  claim 13 , wherein the protein is a sialidase fusion protein. 
     
     
         18 . The method of  claim 17 , wherein the sequence of the sialidase fusion protein comprises the sequence of amino acids set forth in SEQ ID NO:17. 
     
     
         19 . The method of  claim 13 , wherein the temperature is between about 4° C. to about −45° C. 
     
     
         20 . The method of  claim 13 , further comprising adding an active agent to step a) or to a suspension of the formed microspheres, wherein the active agent is selected from among antidiabetics, anticonvulsants, analgesics, antiparkinsons, anti-inflammatories, calcium antagonists, anesthetics, antimicrobials, antimalarials, antiparasitics, antihypertensives, antihistamines, antipyretics, alpha-adrenergic agonists, alpha-blockers, biocides, bactericides, bronchial dilators, beta-adrenergic blocking drugs, contraceptives, cardiovascular drugs, calcium channel inhibitors, depressants, diuretics, electrolytes, enzymes, hormones, hypoglycemics, hyperglycemics, muscle contractants, muscle relaxants, antineoplastics, glycoproteins, nucleoproteins, lipoproteins, ophthalmics, sedatives, steroids, sympathomimetics, parasympathomimetics, tranquilizers, vaccines, vitamins, minerals, nonsteroidal anti-inflammatory drugs, angiotensin converting enzymes, polynucleotides, polypeptides, polysaccharides and nutritional supplements; whereby the microspheres comprise the active agent. 
     
     
         21 . The method of  claim 13 , wherein the amount of organic solvent added is from about 0.1% to about 50% v/v. 
     
     
         22 . The method of  claim 21 , wherein the organic solvent is present in the mixture at about 1% to about 30% v/v. 
     
     
         23 . The method of  claim 13 , wherein the size of the microparticles is from about 0.5 μm to about 5.0 μm. 
     
     
         24 . A method of making protein-based microparticles, comprising:
 a) providing a solution comprising a protein, a counterion, an aqueous solvent and an organic solvent;   b) providing the solution at or cooling the solution to a temperature below about 25° C., whereby a composition containing microparticles comprising the protein is formed; and   c) without any other separation and/or washing steps, isolating the microparticles from unincorporated aqueous solvent and organic solvent by lyophilization.   
     
     
         25 . The method of  claim 24 , wherein the organic solvent is selected from among methanol, ethanol, 1-propanol, isopropanol, tert-butyl alcohol, butanol, acetone, acetonitrile, acetic acid, ethyl acetate, methyl ethyl ketone, methyl t-butyl ether, and dimethyl sulfoxide. 
     
     
         26 . The method of  claim 24 , wherein the counterion is an anionic compound. 
     
     
         27 . The method of  claim 26 , wherein the counterion is selected from among citrate, phosphate, sulfate, pivalate, rubidium, bromine, perchlorate, triethylamine, glycine, itaconate, and arginine, and any salt, acid, or base form thereof. 
     
     
         28 . The method of  claim 24 , wherein the protein is a sialidase fusion protein. 
     
     
         29 . The method of  claim 28 , wherein the sequence of the sialidase fusion protein comprises the sequence of amino acids set forth in SEQ ID NO:17. 
     
     
         30 . The method of  claim 24 , wherein the temperature is between about 4° C. to about −45° C. 
     
     
         31 . The method of  claim 24 , further comprising adding an active agent to step a) or to a suspension of the formed microspheres, wherein the active agent is selected from among antidiabetics, anticonvulsants, analgesics, antiparkinsons, anti-inflammatories, calcium antagonists, anesthetics, antimicrobials, antimalarials, antiparasitics, antihypertensives, antihistamines, antipyretics, alpha-adrenergic agonists, alpha-blockers, biocides, bactericides, bronchial dilators, beta-adrenergic blocking drugs, contraceptives, cardiovascular drugs, calcium channel inhibitors, depressants, diuretics, electrolytes, enzymes, hormones, hypoglycemics, hyperglycemics, muscle contractants, muscle relaxants, antineoplastics, glycoproteins, nucleoproteins, lipoproteins, ophthalmics, sedatives, steroids, sympathomimetics, parasympathomimetics, tranquilizers, vaccines, vitamins, minerals, nonsteroidal anti-inflammatory drugs, angiotensin converting enzymes, polynucleotides, polypeptides, polysaccharides and nutritional supplements; whereby the microspheres comprise the active agent. 
     
     
         32 . The method of  claim 24 , wherein the organic solvent is present in the mixture at about 0.1% to about 50% v/v. 
     
     
         33 . The method of  claim 32 , wherein the organic solvent is present in the mixture at about 1% to about 30% v/v. 
     
     
         34 . The method of  claim 24 , wherein the size of the microparticles is from about 0.5 μm to about 5.0 μm. 
     
     
         35 . A method of making a protein-based composition, comprising:
 a) providing a solution comprising a protein, a counterion, an aqueous solvent and an organic solvent;   b) providing the solution at or cooling the solution to a temperature below about 25° C., whereby a composition containing microparticles comprising the protein is formed; and   c) without any other separation and/or washing steps, isolating the microparticles from the unincorporated reagents by lyophilization.   
     
     
         36 . A method of making protein-based microparticles, comprising:
 a) providing a solution comprising a protein, a counterion, an aqueous solvent and an organic solvent, wherein the organic solvent is selected from among methanol, ethanol, 1-propanol, isopropanol, tert-butyl alcohol, butanol, acetone, acetonitrile, acetic acid, ethyl acetate, methyl ethyl ketone, methyl t-butyl ether, and dimethyl sulfoxide, and the counterion is selected from among citrate, phosphate, sulfate, pivalate, rubidium, bromine, perchlorate, triethylamine, glycine, itaconate, and arginine, and any salt, acid, or base form thereof; and   b) providing the solution at or cooling the solution to a temperature below about 25° C., whereby microparticles comprising the protein are formed.   
     
     
         37 . The method of  claim 36 , wherein the protein is a sialidase fusion protein. 
     
     
         38 . The method of  claim 37 , wherein the sequence of the sialidase fusion protein comprises the sequence of amino acids set forth in SEQ ID NO:17. 
     
     
         39 . The method of  claim 36 , wherein the temperature is between about 4° C. to about −45° C. 
     
     
         40 . The method of  claim 36 , further comprising adding an active agent to step a) or to a suspension of the formed microspheres, wherein the active agent is selected from among antidiabetics, anticonvulsants, analgesics, antiparkinsons, anti-inflammatories, calcium antagonists, anesthetics, antimicrobials, antimalarials, antiparasitics, antihypertensives, antihistamines, antipyretics, alpha-adrenergic agonists, alpha-blockers, biocides, bactericides, bronchial dilators, beta-adrenergic blocking drugs, contraceptives, cardiovascular drugs, calcium channel inhibitors, depressants, diuretics, electrolytes, enzymes, hormones, hypoglycemics, hyperglycemics, muscle contractants, muscle relaxants, antineoplastics, glycoproteins, nucleoproteins, lipoproteins, ophthalmics, sedatives, steroids, sympathomimetics, parasympathomimetics, tranquilizers, vaccines, vitamins, minerals, nonsteroidal anti-inflammatory drugs, angiotensin converting enzymes, polynucleotides, polypeptides, polysaccharides and nutritional supplements; whereby the microspheres comprise the active agent. 
     
     
         41 . The method of  claim 36 , wherein the organic solvent is present in the mixture at about 0.1% to about 50% v/v. 
     
     
         42 . The method of  claim 41 , wherein the organic solvent is present in the mixture at about 1% to about 30% v/v. 
     
     
         43 . The method of  claim 42 , wherein the size of the microparticles is from about 0.5 μm to about 5.0 μm. 
     
     
         44 . A method of making protein-based microparticles, comprising:
 a) providing a solution comprising a protein, a counterion, an aqueous solvent and one or more organic solvents, wherein the total amount of organic solvent(s) in the resulting solution is 50% or less v/v and each organic solvent present in the solution is volatile and none are a polymer; and   b) providing the solution at or cooling the solution to a temperature below about 25° C., whereby microparticles comprising the protein are formed.   
     
     
         45 . The method of  claim 44 , wherein the organic solvent is selected from among methanol, ethanol, 1-propanol, isopropanol, tert-butyl alcohol, butanol, acetone, acetonitrile, acetic acid, ethyl acetate, methyl ethyl ketone, methyl t-butyl ether, and dimethyl sulfoxide. 
     
     
         46 . The method of  claim 44 , wherein the counterion is an anionic compound. 
     
     
         47 . The method of  claim 46 , wherein the counterion is selected from among citrate, phosphate, sulfate, pivalate, rubidium, bromine, perchlorate, triethylamine, glycine, itaconate, and arginine, and any salt, acid, or base form thereof. 
     
     
         48 . The method of  claim 44 , wherein the protein is a sialidase fusion protein. 
     
     
         49 . The method of  claim 48 , wherein the sequence of the sialidase fusion protein comprises the sequence of amino acids set forth in SEQ ID NO:17. 
     
     
         50 . The method of  claim 44 , wherein the temperature is between about 4° C. to about −45° C. 
     
     
         51 . The method of  claim 44 , further comprising adding an active agent to step a) or to a suspension of the formed microspheres, wherein the active agent is selected from among antidiabetics, anticonvulsants, analgesics, antiparkinsons, anti-inflammatories, calcium antagonists, anesthetics, antimicrobials, antimalarials, antiparasitics, antihypertensives, antihistamines, antipyretics, alpha-adrenergic agonists, alpha-blockers, biocides, bactericides, bronchial dilators, beta-adrenergic blocking drugs, contraceptives, cardiovascular drugs, calcium channel inhibitors, depressants, diuretics, electrolytes, enzymes, hormones, hypoglycemics, hyperglycemics, muscle contractants, muscle relaxants, antineoplastics, glycoproteins, nucleoproteins, lipoproteins, ophthalmics, sedatives, steroids, sympathomimetics, parasympathomimetics, tranquilizers, vaccines, vitamins, minerals, nonsteroidal anti-inflammatory drugs, angiotensin converting enzymes, polynucleotides, polypeptides, polysaccharides and nutritional supplements; whereby the microspheres comprise the active agent. 
     
     
         52 . The method of  claim 44 , wherein the organic solvent is present in the mixture at about 0.1% to about 50% v/v. 
     
     
         53 . The method of  claim 52 , wherein the organic solvent is present in the mixture at about 1% to about 30% v/v. 
     
     
         54 . The method of  claim 44 , wherein the concentration of counterion added is from about 0.1 mM to about 100 mM. 
     
     
         55 . The method of  claim 44 , wherein the concentration of counterion added is from about 0.1 mM to about 40 mM. 
     
     
         56 . The method of  claim 44 , wherein the concentration of counterion added is from about 0.1 mM to about 15 mM. 
     
     
         57 . The method of  claim 54 , wherein the concentration of counterion added is from about 0.2 mM to about 50 mM. 
     
     
         58 . The method of  claim 57 , wherein the concentration of counterion added is from about 0.5 mM to about 20 mM. 
     
     
         59 . The method of  claim 44 , wherein the size of the resulting microparticles is from about 0.001 μm to about 50 μm. 
     
     
         60 . The method of  claim 44 , wherein the size of the resulting microparticles is from about 0.3 μm to about 30 μm. 
     
     
         61 . The method of  claim 44 , wherein the size of the resulting microparticles is from about 0.5 μm to about 10 μm. 
     
     
         62 . The method of  claim 44 , wherein the size of the resulting microparticles is from about 0.5 μm to about 6.5 μm. 
     
     
         63 . A composition, comprising microparticles formed according to the method of  claim 44 . 
     
     
         64 . The composition of  claim 63 , wherein the microparticles comprise a counterion selected from among citrate, phosphate, sulfate, pivalate, rubidium, bromine, perchlorate, triethylamine, glycine, itaconate, and arginine, and any salt, acid, or base form thereof. 
     
     
         65 . The composition of  claim 64 , wherein the amount of counterion in the microparticles is from about 0.5% w/w to about 5% w/w. 
     
     
         66 . The composition of  claim 64 , wherein the protein is a sialidase fusion protein comprising a sialidase catalytic domain and a glycosaminoglycan (GAG)-binding domain, wherein the catalytic domain of the sialidase is the only portion of the sialidase in the sialidase fusion protein. 
     
     
         67 . The composition of  claim 66 , wherein the sialidase is  Actinomyces viscosus  sialidase. 
     
     
         68 . The composition of  claim 66 , wherein the GAG-binding domain is selected from among the GAG-binding domain of human platelet factor 4 (SEQ ID NO:3), the GAG-binding domain of human interleukin 8 (SEQ ID NO:4), the GAG-binding domain of human antithrombin 111 (SEQ ID NO:5), the GAG-binding domain of human apoprotein E (SEQ ID NO:6), the GAG-binding domain of human angio-associated migratory protein (SEQ ID NO:7) and the GAG-binding domain of human amphiregulin (SEQ ID NO:8). 
     
     
         69 . The composition of  claim 68 , wherein the GAG-binding domain is that of human amphiregulin (SEQ ID NO:8). 
     
     
         70 . The composition of  claim 69 , wherein the amino acid sequence of the sialidase fusion protein comprises the sequence of amino acid residues set forth in SEQ ID NO:17. 
     
     
         71 . The composition of  claim 63 , wherein the size of the microparticles is from about 0.001 μm to about 50 μm. 
     
     
         72 . The composition of  claim 71 , wherein the size of the resulting microparticles is from about 0.3 μm to about 30 μm. 
     
     
         73 . The composition of  claim 72 , wherein the size of the resulting microparticles is from about 0.5 μm to about 10 μm. 
     
     
         74 . A composition comprising microparticles of a protein and a counterion, wherein:
 (a) the size of the microparticles is from about 0.5 μm to about 10 μm; and   (b) the counterion is selected from among citrate, phosphate, sulfate, pivalate, rubidium, bromine, perchlorate, triethylamine, glycine, itaconate, and arginine, and any salt, acid, or base form thereof.   
     
     
         75 . The composition of  claim 74 , wherein the protein is a sialidase fusion protein comprising a sialidase catalytic domain and a glycosaminoglycan (GAG)-binding domain, wherein the catalytic domain of the sialidase is the only portion of the sialidase in the sialidase fusion protein.