# Patent

## Title
Methods of processing compositions containing microparticles

## Abstract

Methods for processing microparticles involve providing a composition comprising a plurality of solid microparticles and at least one non-volatile material, providing a non-solvent, and exposing the composition to the non-solvent to remove at least a portion of the non-volatile material from the composition while retaining at least the microparticles.

## Background
BACKGROUND OF THE INVENTION
1. Technical Field
The present disclosure relates to compositions and formulations containing microparticles and to methods of processing such compositions and formulations.
2. Description of Related Technology
Microparticles, microspheres, and microcapsules, referred to herein collectively as “microparticles,” are solid or semi-solid particles having a diameter of less than one millimeter, more preferably less than 100 microns, which can be formed of a variety of materials, including but not limited to various polymers and proteins. Microparticles have been used in many different applications, primarily separations, diagnostics, and drug delivery.
The most well known examples of microparticles used in separations techniques are those which are formed of polymers of either synthetic or protein origin, such as polyacrylamide, hydroxyapatite, or agarose. These polymeric microparticles are commonly used to separate molecules such as proteins based on molecular weight and/or ionic charge, or by interaction with molecules chemically coupled to the microparticles.
In the diagnostic area, spherical beads or particles have been commercially available as a tool for biochemists for many years. For example, microparticles have been derivatized with an enzyme, a substrate for an enzyme, or a labeled antibody, and then interacted with a molecule to be detected, either directly or indirectly. A number of derivatized beads are commercially available, all with various constituents and sizes.
In the controlled drug delivery area, molecules have been encapsulated within microparticles or incorporated into a matrix, to provide controlled release of the molecules. A number of different techniques have been used to make such microparticles from various polymers including phase separation, solvent evaporation, emulsification, and spray drying. Generally, the polymers form the supporting structure of the microspheres, and the drug or molecule of interest is incorporated into the supporting structure. Exemplary polymers used for the formation of microspheres include homopolymers and copolymers of lactic acid and glycolic acid (PLGA), block copolymers, and polyphosphazenes.
SUMMARY OF THE INVENTION
In one embodiment, the methods of processing microparticles involve providing a composition comprising a plurality of solid microparticles and at least one non-volatile material, providing a non-solvent comprising at least one non-aqueous liquid and at least one aqueous liquid, exposing the composition to the non-solvent to form a mixture containing one or more liquid phases and the solid microparticles, and removing at least a portion of the resulting one or more liquid phases while retaining at least the microparticles, thereby removing at least a portion of the non-volatile material from the composition, wherein the non-volatile material is more soluble in the non-solvent than are the microparticles, and the non-solvent is free of primary, linear alcohols and halogenated solvents.
In another embodiment, the methods of processing microparticles involve providing a composition comprising a plurality of solid microparticles and at least one non-volatile material, providing a non-solvent comprising 2-methyl-2-propanol, exposing the composition to the non-solvent to form a mixture containing one or more liquid phases and the solid microparticles, and removing at least a portion of the resulting one or more liquid phases while retaining at least the microparticles, thereby removing at least a portion of the non-volatile material from the composition, wherein the non-volatile material is more soluble in the non-solvent than are the microparticles.
In a further embodiment, the methods of processing microparticles involve providing a composition comprising a plurality of solid microparticles and at least one non-volatile material, providing a non-solvent comprising 2-methyl-2-propanol, exposing the composition to the non-solvent to form a mixture containing one or more liquid phases and the solid microparticles, and removing at least a portion of the resulting one or more liquid phases while retaining at least the microparticles, thereby removing at least a portion of the non-volatile material from the composition, wherein the non-volatile material is more soluble in the non-solvent than are the microparticles.


DETAILED DESCRIPTION
The present disclosure relates to compositions and formulations containing microparticles and to methods of processing such compositions and formulations. In accordance with the methods, microparticles are separated from reaction/incubation media so that the microparticles can be collected and/or incorporated into compositions and formulations suitable for drug delivery, diagnostic, separations, and other applications.
The disclosed methods are advantageous for a number of reasons including but not limited to the obtained microparticles can be substantially free of residual polymer(s), solvent(s), and/or excipient(s) added during mi crop article formation. Accordingly, the disclosed methods facilitate production of microparticles comprising substantially 100 wt. % active agent(s) and/or macromolecule carrier(s), which are substantially free of residual polymer(s), solvent(s), and/or excipient(s). Furthermore, compositions in the form of multi-phasic dispersions can be processed; as the microparticles can be formed in dispersions, the disclosed methods can facilitate increased manufacturing efficiencies.
Unless otherwise defined herein, scientific and technical terminologies employed in the present disclosure shall have the meanings that are commonly understood and used by one of ordinary skill in the art. Unless otherwise required by context, it will be understood that singular terms shall include plural forms of the same and plural terms shall include the singular. Specifically, as used herein and in the claims, the singular forms “a” and “an” include the plural reference unless the context clearly indicates otherwise. Thus, for example, the reference to a microparticle is a reference to one such microparticle or a plurality of such microparticles, including equivalents thereof known to one skilled in the art. Also, as used herein and in the claims, the terms “at least one” and “one or more” have the same meaning and include one, two, three or more. The following terms, unless otherwise indicated, shall be understood to have the following meanings when used in the context of the present disclosure.
“Dispersion” refers to a mixture of matters having at least one dispersed or discontinuous phase (optionally, being finely divided, such as in the form of solid microparticles) present in a solid or non-solid continuous phase (e.g., fluidic, liquid, aqueous, organic, gaseous). Representative examples of dispersions in accordance with the disclosure include solid in solid, solid in liquid, solid in gas, and the like. A dispersion can be substantially homogenous or non-homogenous. A suspension is a particular dispersion in which the discontinuous solid phase (such as microparticles) can remain stably suspended (substantially free of sedimentation) in the continuous phase for extended periods of time (for example, at least 5 seconds, 10 seconds, or 30 seconds, e.g., minutes, hours, days, weeks, months, or even one year or more). The dispersion is typically free of water-immiscible liquids, but multi-phasic dispersions are also possible. “Multi-phasic dispersions” are dispersions having at least two phases, for example, three or even more phases. In one example, such dispersions may comprise two immiscible solvents or solvent systems in addition to a dispersed phase.
“Microparticle” refers to a solid particulate (including substantially solid or semi-solid, but excluding gel, liquid and gas) having an average geometric particle size (sometimes referred to as diameter) of less than about 1 mm, for example, less than about 200 microns, less than about 100 microns, less than about 10 microns, less than about 1 micron, less than about 100 nm, less than about 10 nm, greater than about 0.1 nm, greater than about 1 nm, and ranges between these values. Thus, suitable ranges for average geometric particle size include about 0.1 nm to about 1 mm, about 1 nm to about 1 mm, about 10 nm to about I mm, about 100 nm to about 1 mm, about 1 micron to about 1 mm, about 10 microns to about 1 mm, about 100 microns to about 1 mm, about 200 microns to about 1 mm, about 0.1 nm to about 200 microns, about 1 nm to about 200 microns, about 10 nm to about 200 microns, about 100 nm to about 200 microns, about 1 micron to about 200 microns, about 10 microns to about 200 microns, about 100 microns to about 200 microns, about 0.1 nm to about 100 microns, about I nm to about 100 microns, about 10 nm to about 100 microns, about 100 nm to about 100 microns, about 1 micron to about 100 microns, about 10 microns to about 100 microns, about 0.1 nm to about 10 microns, about 1 nm to about 10 microns, about 10 nm to about 10 microns, about 100 nm to about 10 microns, about 1 micron to about 10 microns, about 0.1 nm to about 1 micron, about 1 nm to about 1 micron, about 10 nm to about 1 micron, about 100 nm to about 1 micron, about 0.1 nm to about 100 nm, about 1 nm to about 100 nm, about 10 nm to about 100 nm, about 0.1 nm to about 10 nm, about 1 nm to about 10 nm, and/or about 0.1 nm to about 1 nm. Average geometric particle size can be measured by dynamic light scattering methods (such as photocorrelation spectroscopy, laser diffraction, low-angle laser light scattering (LALLS), medium-angle laser light scattering (MALLS)), light obscuration methods (such as Coulter analysis method), or other methods (such as rheology, light or electron microscopy). Microparticles for pulmonary delivery have an aerodynamic particle size as determined by time of flight measurements or Andersen Cascade Impactor measurements. Microparticles having a spherical shape are sometimes referred to as microspheres and nanospheres. Microparticles having an encapsulated structure are sometimes referred to as microcapsules and nanocapsules. Microparticles can be porous, for example, having one or more internal voids and/or cavities. Other microparticles are non-porous and/or are free of such voids or cavities. Microparticles are formed from, in part or in whole, one or more materials including but not limited to active agents, carriers, polymers, complexing agents, stabilizing agents, excipients, ions, moisture, residual solvents, impurities, by-products, and/or manufacturing-related compounds. Microparticles can be crystalline, amorphous, microcrystalline, nanocrystalline, or a combination thereof.
“Active agent” refers to naturally occurring, recombinant, synthetic, or semi-synthetic materials (e.g., compounds, fermentates, extracts, cellular structures) capable of eliciting, directly or indirectly, one or more physical, chemical, and/or biological effects, in vitro and/or in vivo. The active agent can be capable of preventing, alleviating, treating, and/or curing abnormal and/or pathological conditions of a living body, such as by destroying a parasitic organism, or by limiting the effect of a disease or abnormality by materially altering the physiology of the host or parasite. The active agent can be capable of maintaining, increasing, decreasing, limiting, or destroying a physiological body function. The active agent can be capable of diagnosing a physiological condition or state by an in vitro and/or in vivo test. The active agent can be capable of controlling or protecting an environment or living body by attracting, disabling, inhibiting, killing, modifying, repelling and/or retarding an animal or microorganism. The active agent can be capable of otherwise treating (such as deodorizing, protecting, adorning, grooming) a body. Depending on the effect and/or its application, the active agent can further be referred to as a bioactive agent, a pharmaceutical agent (such as a prophylactic agent, a therapeutic agent), a diagnostic agent, a nutritional supplement, and/or a cosmetic agent, and includes, without limitation, examples such as prodrugs, affinity molecules, synthetic organic molecules, polymers, molecules with a molecular weight of 2 kDa or less (such as those having a molecular weight of less than about 1.5 kDa, or less than about 1 kDa), macromolecules (such as those having a molecular weight of greater than about 2 kDa, for example, greater than about 5 kDa or from about 2 kDa to about 5 kDa), proteinaceous compounds, peptides, vitamins, steroids, steroid analogs, lipids, nucleic acids, carbohydrates, precursors thereof, and derivatives thereof. Active agents can be ionic or non-ionic, can be neutral, positively charged, negatively charged, or zwitterionic, and can be used singly or in combination of two or more thereof. Active agents can be water-insoluble or water-soluble. Active agents can have an isoelectric point of 7.0 or greater, or less than 7.0.
“Proteinaceous compounds” refer to natural, synthetic, semi-synthetic, or recombinant compounds of or related structurally and/or functionally to proteins, such as those containing or consisting essentially of α-amino acids covalently associated through peptide linkages. Non-limiting proteinaceous compounds include globular proteins (e.g., albumins, globulins, histones), fibrous proteins (e.g., collagens, elastins, keratins), compound proteins (including those containing one or more non-peptide components, e.g., glycoproteins, nucleoproteins, mucoproteins, lipoproteins, metalloproteins), therapeutic proteins, fusion proteins, receptors, antigens (such as synthetic or recombinant antigens), viral surface proteins, hormones and hormone analogs, antibodies (such as monoclonal or polyclonal antibodies), enzymes, Fab fragments, cyclic peptides, linear peptides, and the like. Non-limiting therapeutic proteins include bone morphogenic proteins, drug resistance proteins, toxoids, erythropoietins, proteins of the blood clotting cascade (e.g., Factor VII, Factor VIII, Factor IX, et al.), subtilisin, ovalbumin, alpha-1-antitryp sin (AAT), DNase, superoxide dismutase (SOD), lysozymes, ribonucleases, hyaluronidase, collagenase, human growth hormone (hGH), erythropoietin, insulin, insulin-like growth factors, interferons, glatiramer, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, desmopressin, leutinizing hormone release hormone (LHRH) agonists (e.g., leuprolide, goserelin, buserelin, gonadorelin, histrelin, nafarelin, deslorelin, fertirelin, triptorelin), LHRH antagonists, vasopressin, cyclosporine, calcitonin, parathyroid hormone, parathyroid hormone peptides, glucogen-like peptides, and analogs thereof. Proteinaceous compounds may be neutral, positively charged, negatively charged, or zwitterionic, and may be used singly or in combination of two or more thereof
“Nucleic acids” refer to natural, synthetic, semi-synthetic, or recombinant compounds formed at least in part from two or more of the same or different nucleotides, and may be single-stranded or double-stranded. Non-limiting examples of nucleic acids include oligonucleotides (such as those having 20 or less base pairs, e.g., sense, anti-sense, or missense), aptamers, polynucleotides (e.g., sense, anti-sense, or missense), DNA (e.g., sense, anti-sense, or missense), RNA (e.g., sense, anti-sense, or missense), siRNA, nucleotide acid constructs, single-stranded or double-stranded segments thereof, as well as precursors and derivatives thereof (e.g., glycosylated, hyperglycosylated, PEGylated, FITC-labeled, nucleosides, salts thereof). Nucleic acids may be neutral, positively charged, negatively charged, or zwitterionic, and may be used singly or in combination of two or more thereof.
“Macromolecule” refers to a material capable of providing a three-dimensional (e.g., tertiary and/or quaternary) structure, and includes carriers and certain active agents of the present disclosure. Macromolecules typically have a molecular weight of 2 kD or greater, for example, greater than about 5 kD or between 2 kD and 5 kD. Non-limiting macromolecules used to form the microparticles include, inter alia, polymers, copolymers, proteins (e.g., enzymes, recombinant proteins, albumins such as human serum albumin, monoclonal antibodies, polyclonal antibodies, proteinaceous compounds), peptides, lipids, carbohydrates (e.g., monosaccharides, disaccharides, polysaccharides), nucleic acids, vectors (e.g., viruses, viral particles), and complexes and conjugates thereof (e.g., covalent and/or non-covalent associations between two macromolecules such as carbohydrate-protein complexes or conjugates, or between an active agent and a macromolecule such as hapten-protein complexes or conjugates). Macromolecules may be neutral, positively charged, negatively charged, or zwitterionic, and may be used singly or in combination of two or more thereof.
“Carrier” refers to a compound, typically a macromolecule, having a primary function to provide a three-dimensional structure (including tertiary and/or quaternary structure) to the microspheres. The carrier may be unassociated or associated with the active agent (such as conjugates or complexes thereof) in forming microparticles as described above. The carrier may further provide other functions, such as being an active agent, modifying a release profile of the active agent from the microparticle, and/or imparting one or more particular properties to the microparticle (such as contribute at least in part to the net surface charge). In one example, the carrier is a protein (e.g., an albumin such as human serum albumin) having a molecular weight of 1500 Daltons or greater.
“Polymer” or “polymeric” refers to a natural, recombinant, synthetic, or semi-synthetic molecule having in at least one main chain, branch, or ring structure two or more repeating monomer units. Polymers broadly include dimers, trimers, tetramers, oligomers, higher molecular weight polymers, adducts, homopolymers, random copolymers, pseudo-copolymers, statistical copolymers, alternating copolymers, periodic copolymers, bipolymers, terpolymers, quaterpolymers, other forms of copolymers, substituted derivatives thereof, and mixtures thereof. In one aspect, the terms polymer and polymeric refer to molecules having 10 or more repeating monomer units. Polymers can be linear, branched, block, graft, monodisperse, polydisperse, regular, irregular, tactic, isotactic, syndiotactic, stereoregular, atactic, stereoblock, single-strand, double-strand, star, comb, dendritic, and/or ionomeric, can be ionic or non-ionic, can be neutral, positively charged, negatively charged, or zwitterionic, and can be used singly or in combination of two or more thereof.
“Spherical” refers to a geometric shape that is at least “substantially spherical.” “Substantially spherical” means that the ratio of the longest length (i.e., one between two points on the perimeter and passes the geometric center of the shape) to the shortest length on any cross-section that passes through the geometric center is less than about 1.5, such as less than about 1.33, or less than about 1.25. Thus, spherical does not require a line of symmetry. Further, the microparticles can have surface texturing (such as continuous or discrete lines, islands, lattice, indentations, channel openings, protuberances that are small in scale when compared to the overall size of the microparticles) and still be considered spherical. Surface contact between microparticles is minimized when the microparticles are spherical, and thus undesirable agglomeration of the microparticles is typically minimized. In comparison, microparticles that are aspherical crystals or flakes typically display observable agglomeration through ionic and/or non-ionic interactions at relatively large flat surfaces.
“Solid” refers to a state that includes at least substantially solid and/or semi-solid, but excludes liquid and gas.
“Ambient temperature” refers to a temperature of around room temperature, typically in a range of about 20° C. to about 40° C., for example, about 20° C. to about 25° C.
“Formed from” and “formed of” denote open language. As such, it is intended that a composition formed from or formed of a list of recited components be a composition comprising at least these recited components, and can further include other non-recited components during formulation of the composition and/or in the final obtained product.
Unless otherwise expressly specified, all of the numerical ranges, amounts, values and percentages such as those for quantities of materials, times, temperatures, reaction conditions, ratios of amounts, values for molecular weight (whether number average molecular weight Mn or weight average molecular weight Mw), and others disclosed herein should be understood as modified in all instances by the term “about,” if about is not expressly used in combination with said ranges, amounts, values, and percentages herein. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present disclosure and attached claims are approximations that can vary. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, is inherently somewhat uncertain because of the standard deviation found in its respective testing measurement. Furthermore, when numerical ranges of varying scope are set forth herein, it is contemplated that any combination of these values inclusive of the recited values can be used in accordance with the teachings of the disclosure.
Examples provided herein, including those following “such as” and “e.g.,” are considered as illustrative only of various aspects and features of the present disclosure and embodiments thereof, and thus should not alter the scope of any of the referenced terms or phrases. Any suitable equivalents, alternatives, and modifications thereof (including materials, substances, constructions, compositions, formulations, means, methods, conditions, etc.) known and/or available to one skilled in the art can be used or carried out in place of or in combination with those disclosed herein, and are considered to fall within the scope of the present disclosure. Throughout the present disclosure in its entirety, any and all of the one, two, or more features and aspects disclosed herein, explicitly or implicitly, following terms “example”, “examples”, “such as”, “e.g.”, and the likes thereof may be practiced in any combinations of two, three, or more thereof (including their equivalents, alternatives, and modifications), whenever and wherever appropriate as understood by one of ordinary skill in the art. Therefore, specific details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ aspects and features of the present disclosure in virtually any appropriate manner as understood by one of ordinary skill in the art.
Microparticles
Non-limiting microparticles, materials and methods for fabricating microparticles, compositions and formulations containing microparticles, and utilities of such microparticles, compositions, and formulations include those disclosed in U.S. Pat. Nos. 5,525,519, 5,554,730, 5,578,709, 5,599,719, 5,981,719, 6,090,925, 6,268,053, and 6,458, 387, U.S. Publication Nos. 20030059474, 20030064033, 20040043077, 20050048127, 20050142201, 20050142205, 20050142206, 20050147687, 20050170005, 20050233945, 20060018971, 20060024240, 20060024379, 20060260777, 20070092452, 20070207210, and 20070281031, the disclosures of which are herein incorporated by reference in their entirety. Microparticles can have a generally uniform size distribution, such as a monodisperse size distribution, or a polydisperse size distribution, and a generally uniform shape, such as being substantially spherical. One or more characteristics of the microparticles can be adjusted during fabrication by manipulating one or more variables such as, but not limited to, selection of ingredients or combination thereof, concentrations of different ingredients, reaction temperature, reaction time, and/or pH if reaction is taken place in aqueous solution.
Microparticles are suitable for delivering, in vivo, ex vivo, and/or in vitro, one active agent or a combination of two or more active agents with rapid and/or controlled release profiles, and are useful for a wide variety of therapeutic, pharmaceutical, diagnostic, medical, medicinal, cosmetic, nutritional, biocidic, separational, industrial, commercial, and research applications, such as drug delivery, vaccination, gene therapy and histopathological or in vivo tissue or tumor imaging. Microparticles can be formulated for oral, parenteral, mucosal; ophthalmic; intravenous, subcutaneous, subdermal, intradermal, intra-articular, intramuscular, pulmonary (including oral and nasal inhalations), and/or topical administrations to a subject. Intravenous administration includes catheterization and angioplasty.
The microparticles typically contain one or more macromolecules. The one or more macromolecules (typically, one or more bioactive macromolecules and/or one or more carrier macromolecules) may comprise at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 98%, and up to 100%, or less than 100%, by weight and/or volume of the microparticle, or be present in a range between any two of such values. It will be understood by those skilled in the art that the macromolecule can be a portion (e.g., fragment, segment, subunit) of another larger macromolecule. It will be further understood that macromolecules include affinity molecules, which can be, for example, the receptor or ligand portions of a receptor-ligand interaction. Non-limiting examples of ligands include viruses, bacteria, polysaccharides, or toxins that act as antigens to generate immune responses when administered to an animal and cause the production of antibodies.
One or more ingredients other than the macromolecules described above and the active agents described below including but not limited to polymers, complexing agents, stabilizing agents, excipients, ions, moisture, residual solvents, impurities, by-products, may be present in the microparticle at a quantity of 50% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 2% or less, or greater than 0%, by weight and/or volume of the microparticle, or in a range between any two of such values. Additionally, any ingredients present in the reaction/incubation medium (e.g., such as non-volatile materials) during the formation of the microparticles can be substantially removed from and thus absent in the resulting microparticles. Immediately or at a later stage following their formation (which may or may not be in-situ), the microparticles may be dispersed (e.g., as colloids or suspensions) in a continuous solid phase (e.g., a frozen solid comprising the dispersion) or in a non-solid phase (e.g., a flowable medium, such as the reaction/incubation medium in which the microparticles are formed, or a washing medium).
The microparticles may have a density substantially the same as or different from (such as greater than or less than) that of the continuous phase (measured at the same temperature, such as ambient temperature). Densities of the microparticles, and the continuous phase equal their respective weight divided by their respective volume. The microparticles may have a density less than, equal to, or greater than values such as 0.5 g/cm3, 0.8 g/cm3, 0.95 g/cm3, 1.0 g/cm3, 1.05 g/cm3, 1.1 g cm3, 1.3 g/cm3, 1.35 g/cm3, 1.5 g/cm3, and 1.9 g/cm3, or in a range between any two of such values, such as between 1.0 g/cm3 and 1.5 g/cm3 or between 1.2 g/cm3 and 1.5 g/cm3. Density of the microparticles may be measured by helium pycnometry at ambient temperature, by density-gradient techniques (e.g., using centrifugation or ultracentrifugation) using suitable gradient medium (e.g., salts of alkali metals such as NaCl, NaBr, NaI, KBr, CsF, CsCl, CsBr, cesium sulfate, cesium acetate, cesium trifluoroacetate, RbCl, and potassium tartrate; neutral, water-soluble molecules such as sucrose with optional addition of glucose, glycerol, or mineral oil; hydrophilic macromolecules such as dextran, sucrose-epichlorohydrin copolymer, and bovine serum albumin; other synthetic molecules such as sodium or methyl glucamine salts of triiodobenzoic acid and of metrizoic acid, and metrizamide), and other known methods. Standard methods involving density-gradient techniques include ASTM D1505-03, ASTM D1505-98, and ISO 1183-2.
Active Agents
One or more active agents are typically covalently and/or non-covalently associated with, and/or entrapped by, at least a portion (e.g., the center or core, one or more specifically or randomly distributed compartments, inner and/or outer surfaces) of the microparticle. For example, the one or more active agents may be covalently and/or non-covalently associated with, and/or entrapped by, at least a portion or substantially all of one or more macromolecules (e.g., bioactive macromolecules and/or carrier macromolecules) and/or one or more other ingredients (e.g., with one or more polymers, as complexes or conjugates thereof). Alternatively, the macromolecule itself can comprise an active agent. In both instances, the microparticles can comprise a bioactive macromolecule on an outer surface thereof.
The active agent may be a pharmaceutical agent. Depending on its effect and/or application, the pharmaceutical agent includes, without limitation, adjuvants, adrenergic agents, adrenergic blocking agents, adrenocorticoids, adrenolytics, adrenomimetics, alkaloids, alkylating agents, allosteric inhibitors, anabolic steroids, analeptics, analgesics, anesthetics, anorexiants, antacids, anti-allergic agents, antiangiogenesis agents, anti-arrhythmic agents, anti-bacterial agents, antibiotics, antibodies, anticancer agents such as paclitaxel and derivative compounds, anticholinergic agents, anticholinesterases, anticoagulants, anticonvulsants, antidementia agents, antidepressants, antidiabetic agents, antidiarrheals, antidotes, antiepileptics, antifolics, antifungals, antigens, antihelmintics, antihistamines, antihyperlipidemics, antihypertensive agents, anti-infective agents, anti-inflammatory agents, antimalarials, antimetabolites, antimuscarinic agents, antimycobacterial agents, antineoplastic agents, antiosteoporosis agents, antipathogen agents, antiprotozoal agents, adhesion molecules, antipyretics, antirheumatic agents, antiseptics, antithyroid agents, antiulcer agents, antiviral agents, anxiolytic sedatives, astringents, beta-adrenoceptor blocking agents, biocides, blood clotting factors, calcitonin, cardiotonics, chemotherapeutics, cholesterol lowering agents, cofactors, corticosteroids, cough suppressants, cytokines, diuretics, dopaminergics, estrogen receptor modulators, enzymes and cofactors thereof, enzyme inhibitors, growth differentiation factors, growth factors, hematological agents, hematopoietics, hemoglobin modifiers, hemostatics, hormones and hormone analogs, hypnotics, hypotensive diuretics, immunological agents, immunostimulants, immunosuppressants, inhibitors, ligands, lipid regulating agents, lymphokines, muscarinics, muscle relaxants, neural blocking agents, neurotropic agents, parasympathomimetics, parathyroid hormone, promoters, prostaglandins, psychotherapeutic agents, psychotropic agents, radio-pharmaceuticals, receptors, sedatives, sex hormones, sterilants, stimulants, thrombopoietics, trophic factors, sympathomimetics, thyroid agents, vaccines, vasodilators, vitamins, xanthines, as well as conjugates, complexes, precursors, and metabolites thereof. The active agent may be used individually or in combinations of two or more thereof. In one example, the active agent is a prophylactic and/or therapeutic agent that includes, but is not limited to, peptides, carbohydrates, nucleic acids, other compounds, precursors and derivatives thereof, and combinations of two or more thereof. In one aspect, the active agent is a pharmaceutical agent that is conventionally referred to as a small molecule.
The active agent may be a bioactive active agent, for example, a bioactive macromolecule, such as a protein (including the proteinaceous compounds described above), a polypeptide, a carbohydrate, a polynucleotide, a vector (e.g., a virus or viral particle), or a nucleic acid, or a combination of two or more thereof The macromolecule can be natural or synthetic. Exemplary proteins include monoclonal antibodies, polyclonal antibodies. The protein can also be any known therapeutic proteins isolated from natural sources or produced by synthetic or recombinant methods. Examples of therapeutic proteins include, but are not limited to, proteins of the blood clotting cascade (e.g., Factor VII, Factor VIII, Factor IX, et al.), subtilisin, ovalbumin, alpha-1-antitrypsin (AAT), DNase, superoxide dismutase (SOD), lysozyme, ribonuclease, hyaluronidase, collagenase, growth hormone, erythropoietin, insulin-like growth factors or their analogs, interferons, glatiramer, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, antibodies, PEGylated proteins, glycosylated or hyperglycosylated proteins, desmopressin, LHRH agonists such as: leuprolide, goserelin, nafarelin, buserelin; LHRH antagonists, vasopressin, cyclosporine, calcitonin, parathyroid hormone, parathyroid hormone peptides and insulin.
The active agent may be a cosmetic agent. Non-limiting examples of cosmetic agents include emollients, humectants, free radical inhibitors, anti-inflammatory agents, vitamins, depigmenting agents, anti-acne agents, antiseborrhoeics, keratolytics, slimming agents, skin coloring agents, and sunscreen agents. Non-limiting compounds useful as cosmetic agents include linoleic acid, retinol, retinoic acid, ascorbic acid alkyl esters, polyunsaturated fatty acids, nicotinic esters, tocopherol nicotinate, unsaponifiables of rice, soybean or shea, ceramides, hydroxy acids such as glycolic acid, selenium derivatives, antioxidants, beta-carotene, gamma-orizanol, and stearyl glycerate. The cosmetic agents may be commercially available and/or prepared by known techniques. As above, the various active agents may be used individually or in combinations of two or more thereof.
The active agent may be a nutritional supplement. Non-limiting examples of nutritional supplements include proteins, carbohydrates, water-soluble vitamins (e.g., vitamin C, B-complex vitamins, and the like), fat-soluble vitamins (e.g., vitamins A, D, E, K, and the like), and herbal extracts. The nutritional supplements may be commercially available and/or prepared by known techniques. As above, the various active agents may be used individually or in combinations of two or more thereof.
The active agent may be a compound having a molecular weight of 2 kDa or less. Non-limiting examples of such compounds include steroids, beta-agonists, anti-microbial agents, antifungal agents, taxanes (antimitotic and antimicrotubule agents), amino acids, aliphatic compounds, aromatic compounds, and urea compounds. Active agents conventionally known as small molecules (or small organic molecules) are representative active agents having a molecular weight of 2 kDa or less.
The active agent may also be a diagnostic agent. Non-limiting diagnostic agents include x-ray imaging agents and contrast media. Non-limiting examples of x-ray imaging agents include ethyl 3,5-diacetamido-2,4,6-triiodobenzoate (WIN-8883, ethyl ester of diatrazoic acid); 6-ethoxy-6-oxohexyl-3,5-bis(acetamido)-2,4,6-triiodobenzoate (WIN 67722); ethyl-2-(3,5-bis(acetamido)-2,4,6-triiodobenzoyloxy)-butyrate (WIN 16318); ethyl diatrizoxyacetate (WIN 12901); ethyl 2-(3,5-bis(acetamido)-2,4,6-triiodobenzoyloxy)propionate (WIN 16923); N-ethyl 2-(3,5-bis(acetamido)-2,4,6-triiodobenzoyloxy-acetamide (WIN 65312); isopropyl 2-(3,5-bis(acetamido)-2,4,6-triiodobenzoyloxy)acetamide (WIN 12855); diethyl 2-(3,5-bis(acetamido)-2,4,6-triiodobenzoyloxymalonate (WIN 67721); ethyl 2-(3,5-bis(acetamido)-2,4,6-triiodobenzoyloxy)phenyl-acetate (WIN 67585); propanedioic acid, [[3,5-bis(acetylamino)-2,4,5-triodobenzoyl]oxy]bis(1-methyl)ester (WIN 68165); and benzoic acid, 3,5-bis(acetylamino)-2,4,6-triodo-4-(ethyl-3-ethoxy-2-butenoate)ester (WIN 68209). Preferred contrast agents desirably disintegrate relatively rapidly under physiological conditions, thus minimizing any particle associated inflammatory response. Disintegration may result from enzymatic hydrolysis, solubilization of carboxylic acids at physiological pH, or other mechanisms. Thus, poorly soluble iodinated carboxylic acids such as iodipamide, diatrizoic acid, and metrizoic acid, along with hydrolytically labile iodinated species such as WIN 67721, WIN 12901, WIN 68165, and WIN 68209 or others may be preferred.
In one specific embodiment, the active agent may be a therapeutic agent for prevention and/or treatment of pulmonary disorders. Non-limiting examples of such agents include steroids, beta-agonists, anti-fungal agents, anti-microbial compounds, bronchial dialators, anti-asthmatic agents, non-steroidal anti-inflammatory agents (NSAIDS), AAT, and agents to treat cystic fibrosis. Non-limiting examples of steroids for prevention and/or treatment of pulmonary disorders include but are not limited to beclomethasone (such as beclomethasone dipropionate), fluticasone (such as fluticasone propionate), budesonide, estradiol, fludrocortisone, flucinonide, triamcinolone (such as triamcinolone acetonide), flunisolide, and salts thereof. Non-limiting examples of beta-agonists for prevention and/or treatment of pulmonary disorders include salmeterol xinafoate, formoterol fumarate, levo-albuterol, bambuterol, tulobuterol, and salts thereof. Non-limiting examples of anti-fungal agents for prevention and/or treatment of pulmonary disorders include itraconazole, fluconazole, amphotericin B, and salts thereof.
The active agents may be used in a combination of two or more thereof Non-limiting exemplary combinations include a steroid and a beta-agonist, e.g., fluticasone propionate and salmeterol, budesonide and formoterol, etc. Many other viable therapeutically active agent combinations are well known to those of ordinary skill in the art.
Non-Volatile Material
Compositions containing the microparticles can contain at least one non-volatile material, the at least one non-volatile material(s) being different in their chemical structures and/or compositions from that of the one or more macromolecules that form the microparticles.
Generally, the non-volatile materials have a boiling point and/or a flash point greater than about 100° C., greater than about 150° C., and/or greater than about 200° C. The non-volatile materials can be natural, synthetic, semi-synthetic, or recombinant. The one or more non-volatile materials are typically nonionic polymers which can be independently hydrophilic, amphiphilic, aqueous-soluble (e.g., water-soluble), and/or aqueous-miscible (e.g., water-miscible), or salts of such nonionic polymers. The one or more non-volatile materials can beneficially independently or collectively reduce the solubility of one or more macromolecules in the continuous phase, or in the one or more fluids therein. The one or more non-volatile materials, when present in the continuous phase, typically do not covalently and/or ionically interact with, or denature, the one or more macromolecules in the microparticles. Additionally, the one or more non-volatile materials, when present in the continuous phase, typically do not complex, conjugate, aggregate, and/or agglomerate with each other, or otherwise come together, such as via covalent, ionic, and/or other interactions. Further, the one or more non-volatile materials in the continuous phase typically do not undergo gelation (e.g., form a hydrogel), either by themselves or with other ingredients present in the continuous phase. The one or more non-volatile materials independently generally have molecular weights greater than or equal to values such as 200 daltons, 300 daltons, 400 daltons, 600 daltons, 800 daltons, 1,000 daltons, 1,500 daltons, 2,000 daltons, 2,500 daltons, 3,000 daltons, 3,500 daltons, 4,000 daltons, 5,000 daltons, 8,000 daltons, and 10,000 daltons, or up to about 3,000 kilodaltons (kd), or in a range between any two of such values, for example, between 200 daltons and 10,000 daltons, between 1000 daltons and 1500 daltons, between 1000 daltons and 2,000 daltons, between 1000 daltons and 2,500 daltons, between 1000 daltons and 3,000 daltons, between 1000 daltons and 3,500 daltons, between 1000 daltons and 4,000 daltons, between 1000 daltons and 5,000 daltons, between 1000 daltons and 8,000 daltons, between 1000 daltons and 10,000 daltons, between 1,500 daltons and 2,000 daltons, between 1,500 daltons and 2,500 daltons, between 1,500 daltons and 3,000 daltons, between 1,500 daltons and 3,500 daltons, between 1,500 daltons and 4,000 daltons, between 1,500 daltons and 5,000 daltons, between 1,500 daltons and 8,000 daltons, between 1,500 daltons and 10,000 daltons, between 2,000 daltons and 2,500 daltons, between 2,000 daltons and 3,000 daltons, between 2,000 daltons and 3,500 daltons, between 2,000 daltons and 4,000 daltons, between 2,000 daltons and 5,000 daltons, between 2,000 daltons and 8,000 daltons, between 2,000 daltons and 10,000 daltons, etc.
Non-limiting examples of non-volatile materials for the continuous phase include the non-ionic water-soluble and/or water-miscible polymers disclosed in U.S. Pat. Nos. 5,525,519, 5,554,730, 5,578,709, 5,599,719, 5,981,719, 6,090,925, 6,268,053, and 6,458, 387, U.S. Publication Nos. 20030059474, 20030064033, 20040043077, 20050048127, 20050142201, 20050142205, 20050142206, 20050147687, 20050170005, 20050233945, 20060018971, 20060024240, 20060024379, 20060260777, 20070092452, 20070207210, and 20070281031, the disclosures of which are herein incorporated by reference in their entirety. The non-volatile material(s) are typically non-ionic, and can be hydrophilic, amphiphilic, aqueous-soluble, aqueous-miscible, and/or soluble or miscible in an aqueous-soluble or aqueous-miscible fluid at a temperature of 40° C. or below. Non-limiting examples of suitable non-volatile materials may be linear, branched, or cyclic, and include non-ionic polyethers, non-ionic copolyethers, non-ionic polyesters, non-ionic copolyesters, non-ionic polyether-polyester copolymers, non-ionic vinyl polymers, non-ionic pyrrolidone-containing polymers, non-ionic polymeric carbohydrates, derivatives and salts thereof, and combinations of two or more thereof. Non-limiting examples of non-ionic polyethers and non-ionic copolyethers (including copolymers and terpolymers) include but are not limited to hydroxy-terminated polyethers (e.g., polyether alcohols, polyether polyols, ethylene oxide end-capped polyethers other than polyethylene glycols) and alkyl (e.g., methyl, ethyl, propyl, butyl, etc.) end-capped derivatives thereof, such as polyalkylene glycols (e.g., poly-oxy-1,2-alkylene glycols like polyethylene glycols and polypropylene glycols, as well as polytrimethylene ether glycols and polytetramethylene ether glycols), hydroxy-terminated copolyethers (e.g., copolyether alcohols, copolyether polyols, ethylene oxide end-capped copolyethers) and alkyl (e.g., methyl, ethyl, propyl, butyl, etc.) end-capped derivatives thereof, such as block copolyethers of two or more different 1,2-alkylene oxides (e.g., polyoxyethylene-polyoxypropylene copolymers like poloxamers) and copolyethers of one or more 1,2-alkylene oxides and one or more of tetrahydrofuran, tetrahydropyran, and 1,3-propanediol (e.g., (polyethylene glycol)-(polytrimethylene ether glycol) copolymers, (polyethylene glycol)-(polytetramethylene ether glycol) copolymers). Non-limiting examples of non-ionic polyesters and non-ionic copolyesters (including copolymers and terpolymers) include hydroxy-terminated polyesters (e.g., polyester polyols, copolyester polyols, ethylene oxide end-capped or polyoxyethylene-terminated polyesters, and certain silicone polyesters, such as the likes of polyoxyethylene glycerin dicarboxylic acid esters, polyoxyethylenesorbitol dicarboxylic acid esters, polyoxyethylene glycol dicarboxylic acid esters, and polyoxyethylenealkyl esters. Non-limiting examples of non-ionic polyether-polyester copolymers (including terpolymers) include but are not limited to block copolymers of one or more lactones and/or dicarboxylic acids and one or more 1,2-alkylene oxides), esterification derivatives of the non-ionic polyethers and non-ionic copolyethers disclosed herein, and etherification derivatives of the non-ionic polyesters and non-ionic copolyesters disclosed herein, such as (polyethylene glycol)-polycaprolactone block copolymers. Non-limiting examples of non-ionic vinyl polymers (including copolymers and terpolymers) and pyrrolidone-containing non-ionic polymers (including copolymers and terpolymers) include but are not limited to polyvinyl alcohols, homopolymers and copolymers (including terpolymers) of hydroxyalkyl(alk)acrylates (e.g., hydroxyethyl acrylate, hydroxypropyl acrylate, hydroxyethyl methacrylate, hydroxypropyl methacrylate), oligo-oxyalkylene(alk)acrylates (e.g., oligo-oxyethylene acrylates, oligo-oxyethylene methacrylates), and/or alkyl end-capped oligo-oxyalkylene(alk)acrylates (e.g., methyl-capped), polyvinylpyrrolines, and (alkenyl pyrrolidone)-containing homopolymers and copolymers.
Non-limiting examples of nonionic polymeric (including oligomeric) carbohydrates (having a molecular weight of 200 daltons to 5,000,000 daltons, such as 1,000 daltons, 3,000 daltons, 5,000 daltons, 10,000 daltons, 30,000 daltons, 50,000 daltons, 100,000 daltons, 300,000 daltons, 500,000 daltons, 1,000,000 daltons, or 3,000,000 daltons, or in a range between any two of such values) and derivatives thereof include but are not limited to starch, amylopectin (branched polysaccharides), amylose (linear polysaccharides), cellulose, guar gum, guar polysaccharides, xanthan gum, dextrins (e.g., cyclodextrins, maltodextrins), dextrans, polydextroses, gellan gum, pullulan, cellodextrins, beta-glucans, and derivatives thereof, for example, nonionic esters formed by esterification, including but not limited to benzoates and alkanoates such as acetates, propionates, butyrates, and hexanoates; or nonionic ethers formed by etherification such as nonionic starch ethers, nonionic amylopectin ethers, nonionic amylose ethers, nonionic cellulose ethers, nonionic guar ethers, nonionic starch esters, nonionic amylopectin esters, nonionic amylose esters, nonionic cellulose esters, nonionic starch ether esters, nonionic starch ester ethers, nonionic cellulose ether esters, and nonionic cellulose ester ethers. Non-limiting examples of nonionic starch ethers include alkylstarches such as methylstarches, ethylstarches, propylstarches, and butylstarches; hydroxyalkyl starches such as hydroxyethyl starches (e.g., tetrastarch, pentastarch, hetastarch), hydroxypropyl starches, hydroxybutyl starches, and hydroxypentyl starches; as well as alkylhydroxylalkyl starches such as methylhydroxyethyl starches, methylhydroxypropyl starches, and ethylhydroxypropyl starches. Non-limiting examples of nonionic amylopectin ethers and nonionic amylose ethers include hydroxyethyl amylopectins, hydroxypropyl amylopectins, hydroxyethyl amyloses, and hydroxypropyl amyloses. Non-limiting examples of nonionic cellulose ethers include alkylcelluloses such as methylcelluloses, ethylcelluloses, propylcelluloses, isopropylcelluloses, and butylcelluloses; hydroxyalkyl celluloses such as hydroxyethyl celluloses, hydroxypropyl celluloses, hydroxyisopropyl celluloses, hydroxybutyl celluloses, and hydroxypentyl celluloses; as well as alkylhydroxylalkyl celluloses such as methylhydroxyethyl celluloses, methylhydroxypropyl celluloses, methylhydroxybutyl celluloses, ethylhydroxyethyl celluloses, ethylhydroxypropyl celluloses, propylhydroxyethyl celluloses, propylhydroxypropyl celluloses, isopropylhydroxypropyl celluloses, butylhydroxypropyl celluloses, pentylhydroxypropyl celluloses, and hexylhydroxypropyl celluloses. Non-limiting examples of nonionic guar ethers include alkylguar polysaccharides such as methylguar polysaccharides, ethylguar polysaccharides, propylguar polysaccharides, and butylguar polysaccharides; hydroxyalkylguar polysaccharides such as hydroxyethylguar polysaccharides, and hydroxypropylguar polysaccharides; as well as alkylhydroxylalkylguar polysaccharides such as methylhydroxyethylguar polysaccharides, methylhydroxypropylguar polysaccharides, ethylhydroxypropylguar polysaccharides. Other nonionic polymeric carbohydrates include methyl cellulose, hydroxyethyl cellulose, hydroxyethyl methylcellulose, hydroxypropyl methylcellulose, ethylhydroxyethyl cellulose, methylethylhydroxyethyl cellulose, butylglycidyletherhydroxyethyl cellulose, laurylglycidyletherhydroxyethyl cellulose, hydroxymethylhydroxyethyl cellulose, butylglycidylether modified hydroxyethyl cellulose, methylhydroxyethylcellulose, methylhydroxypropyl cellulose, starch esters (e.g. alkyl succinic anhydride modified starchstarch acetates and starch alkenylsuccinates), cellulose esters (cellulose monobutyrates and monopropionates), cellulose ether esters (hydroxyalkyl cellulose-2-hydroxycarboxylic acid esters), poly(3-hydroxyoxetane)s. Non-limiting examples of nonionic polymeric carbohydrate esters include those having a degree of substitution ranging from 0.5 to 1.0, such as from 0.7 to 0.9, and are water-soluble. Ionic salts of the foregoing materials, if capable of being made, may also be used. For example, water-soluble and/or'miscible salts of polysaccharides such as dextran sulfate, dextrin sulfate, and sodium alginate, can also be used.
Non-Solvent
To enable and/or facilitate the separation of the microparticles from the other components of the composition containing same, or at least the one or more non-volatile materials thereof, one or more non-solvents may be used alone or in combination of two or more thereof. The non-solvent components are selected such that the liquid(s) (e.g., water) and/or the non-volatile material(s) (e.g, a polymer) of the composition are more soluble in and/or miscible with the non-solvent than are the microparticles. As such, the one or more non-solvents may be suitable as differential solubility systems to separate (e.g., extract, wash, exclude, displace, remove) such components from the microparticles, using non-limiting techniques such as washing (e.g, centrifugal washing), diafiltration, filtration, dialysis, electrophoresis, or a combination thereof.
Without being limited thereto, the one or more non-solvents may have one or more of the characteristics (e.g., compositions, physical and chemical properties, toxicity) described herein, or a combination of two or more thereof, provided that they are not in conflict with one another. The non-solvent may be a single-component system or a multi-component system (e.g., binary, ternary, quaternary, etc.) containing at least one or more non-aqueous liquids and one or more aqueous liquids. The non-solvent is typically mono-phasic (having a single phase), and not multi-phasic (e.g., emulsions, suspensions).
Generally, the microparticles are substantially insoluble in the non-solvent or at least the non-aqueous liquid therein. More specifically, the microparticles have a solubility in the non-solvent such that no more than 5% by weight of the microparticles are dissolved as the microparticles are dispersed in the non-solvent, for example, less than 3 wt. %, less than 1 wt. %, less than 0.5 wt. %, less than 0.1 wt. %, less than 0.05 wt. %, less than 0.01 wt. %, or a range between two of these values, for example, between 0.01 wt. % and 5 wt. %, between 0.05 wt. % and 5 wt. %, between 0.1 wt. % and 5 wt. %, between 0.5 wt. % and 5 wt. %, between 1 wt. % and 5 wt. %, between 3 wt. % and 5 wt. %, between 0.01 wt. % and 3 wt. %, between 0.05 wt. % and 3 wt. %, between 0.1 wt. % and 3 wt. %, between 0.5 wt. % and 3 wt. %, between 1 wt. % and 3 wt. %, between 0.01 wt. % and 1 wt. %, between 0.05 wt. % and 1 wt. %, between 0.1 wt. % and 1 wt. %, between 0.5 wt. % and 1 wt. %, between 0.01 wt. % and 0.5 wt. %, between 0.05 wt. % and 0.5 wt. %, between 0.1 wt. % and 0.5 wt. %, between 0.01 wt. % and 0.1 wt. %, between 0.05 wt. % and 0.1 wt. %, and/or between 0.01 wt. % and .05 wt. %.
The non-volatile material should be more soluble in the non-solvent than the microparticles. For example, the non-volatile material(s) can have a solubility in the non-solvent of 10% by weight or greater, such as less than, equal to, or greater than values such as 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or in a range between any two of such values. Additionally, the at least one non-volatile material may be entirely miscible with the non-solvent.
The non-solvent or at least the non-aqueous liquid therein generally have an oral LD50 in rats greater than 1.6 g/kg by body weight, such as 1.7 g/kg or greater, 1.8 g/kg or greater, 2 g/kg or greater, 2.5 g/kg or greater, 3 g/kg or greater, 4 g/kg or greater, 5 g/kg or greater, or in a range between two of such values. The non-solvent is usually free of any materials having an oral LD50 in rats of 1.6 g/kg by body weight or less. Additionally or alternatively, the non-solvent or at least the non-aqueous liquid therein may have a permitted daily exposure to human of 50 mg/day or greater.
The non-solvent may have a density at ambient temperature that is less than or equal to values such as 0.8 g/cm3, 0.85 g/cm3, 0.9 g/cm3, 0.95 g/cm3, 1.0 g/cm3, 1.05 g/cm3, or 1.10 g/cm3 or in a range between any two of such values. Density of the non-solvent equals the weight of the non-solvent divided by its volume, as measured at the same temperature. When used for processing of multi-phasic dispersions comprising dispersed and continuous phases, the non-solvent or at least the non-aqueous liquid therein typically have a density less than or equal to that of the continuous phase or the one or more liquids therein, but this need not be the case.
In one aspect, the non-solvent or at least the non-aqueous liquid therein is preferably lyophilizable. For example, the non-solvent or at least the non-aqueous liquid therein may have a boiling point of 100° C. or less, such as being less than or equal to 90° C., 80° C., 70° C., 60° C., 50° C., 40° C., 30° C., 20° C., 10° C., 5° C., 1° C., 0° C., −5° C., −10° C., −20° C., or in a range between any two of such values. Preferably, the boiling point of the non-solvent or at least the non-aqueous liquid therein is lower than the degradation temperature of the microparticles, and/or lower than the denaturation temperature of the macromolecules and/or bioactive agents (e.g., nucleic acids, proteins including proteinaceous compounds, etc.) in the microparticles such that the microspheres can be dried using traditional evaporation techniques without causing substantial degradation and/or denaturation.
The one or more aqueous liquids, when present, may be selected from aqueous liquids such as H2O, D2O, aqueous solutions containing salts, other solutes, and/or buffers. Non-limiting examples of salts include sodium chloride, sodium sulfate, and other halide and sodium salts. Non-limiting examples of other solutes include carbohydrates (e.g., sugars), polyols, surfactants, and other excipients. Non-limiting examples of buffer salts include ammonium acetate and ammonium bicarbonate.
The one or more nonaqueous liquids are present in the non-solvent in an amount sufficient to prevent the microspheres from being readily dissolved (i.e., to keep the microspheres largely intact). For example, the one or more non-aqueous liquids may be present in an amount (by weight or volume) from 40% or greater to 95% or less, such as being less than, equal to, or greater than values such as 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, or in a range between any two of such values, for example, between 40% and 95%, between 40% and 90%, between 40% and 85%, between 40% and 80%, between 40% and 75%, between 40% and 70%, between 40% and 65%, between 40% and 60%, between 40% and 55%, between 40% and 50%, between 40% and 45%, between 45% and 95%, between 45% and 90%, between 45% and 85%, between 45% and 80%, between 45% and 75%, between 45% and 70%, between 45% and 65%, between 45% and 60%, between 45% and 55%, between 45% and 50%, etc.
The aqueous liquids are present in the non-solvent in an amount sufficient to provide the non-solvent with the properties needed to solvate the non-volatile materials, salts, and excipients of the composition. Typically, the aqueous liquid is present in an amount (by weight or volume) of at least 4%, at least 5%, at least 10%, etc. in order to help solvate the non-volatile materials, salts, and excipients. For example, the aqueous liquid may be present in an amount (by weight or volume) from 1% or greater to 50% or less, such as being less than, equal to, or greater than values such as 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or in a range between any two of such values, for example, between 1% and 45%, between 1% and 40%, between 1% and 35%, between 1% and 30%, between 1% and 25%, between 1% and 20%, between 1% and 15%, between 1% and 10%, between 1% and 5%, between 1% and 15%, between 1% and 10%, between 1% and 5%, between 5% and 50%, between 5% and 45%, between 5% and 40%, between 5% and 35%, between 5% and 30%, between 5% and 25%, between 5% and 20%, between 5% and 15%, between 5% and 10%, etc. In some aspects, relatively smaller amounts of the aqueous solvent (e.g., 20% or less, 15% or less, 10% or less, at least 4%, or at least 5%) are generally preferred to minimize the solubility of the microspheres in the non-solvent, but additional amounts of the aqueous solvent can be included provided the microparticles do not have a substantial solubility in the non-solvent.
The one or more non-aqueous liquids may be present in the non-solvent (by weight or volume) from 40% or greater to 95% or less, such as being less than, equal to, or greater than values such as 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or in a range between any two of such values, for example, between 40% and 95%, between 40% and 90%, between 40% and 85%, between 40% and 80%, between 40% and 75%, between 40% and 70%, between 40% and 65%, between 40% and 60%, between 40% and 55%, between 40% and 50%, between 40% and 45%, etc. (by weight or volume). Most frequently, relatively smaller amounts of the aqueous solvent (e.g., 20% or less, 15% or less, 10% or less, at least 4%, or at least 5%) are sufficient to provide the non-solvent with the properties needed to solvate any aqueous components of the continuous phase.
The aqueous liquid(s) can be partially or fully soluble in or miscible with the non-aqueous liquid(s) of the non-solvent. Further, when multi-phasic dispersions are processed, the continuous phase or at least one liquid thereof may beneficially be soluble in or partially or fully miscible with the non-solvent such that a single phase is formed when the dispersion and the non-solvent are combined.
The at least one non-aqueous liquid in the non-solvent is typically aqueous-miscible. The term “aqueous-miscible non-aqueous liquids” refers to non-aqueous liquids that, at a temperature range representative of a chosen operational environment (e.g., 20° C. to 40° C.), are partially (e.g., at least 5% by weight or volume) or substantially (at all proportions) soluble in and/or miscible with water, for example, without resulting in a significant (e.g., 50% or greater) increase in viscosity (such as through a phase separation). The aqueous-miscible non-aqueous liquid(s) may further contain one or more solutes (e.g., salts, sugars, polymers, etc.) and/or miscible non-aqueous liquids therein. The non-aqueous liquid(s) may be anhydrous and/or of low water content (e.g., those having water content of 5% or less by weight or volume, such as 3% or less, 1% or less, 0.5% or less, 0.1% or less).
Non-limiting examples of non-aqueous liquids include those that are polar aprotic or dipolar aprotic, such as ketones, nitriles, esters, aldehydes, and amides; those that are nonpolar aprotic, such as ethers (e.g., methoxylated ethers, alkylated ethers, diethers, triethers, oligo ethers, polyethers, cyclic ethers, crown ethers); tertiary organic liquids (e.g., tertiary alcohols, tertiary acids, tertiary amides); and combinations thereof, for example, such as nitriles+dimethylsulfoxide, nitriles+ethers, nitriles+ketones, tertiary alcohols+(cyclo)alkanes, ketones+tertiary alcohols, nitriles+tertiary alcohols, nitriles+ketones+tertiary alcohols, nitriles+ethers+tertiary alcohols. In one aspect, the non-aqueous liquid is free of primary, linear alcohols (e.g., methanol, ethanol, propanol, butanol, etc.) and halogenated solvents (e.g., dichloromethane, chloroform, trichloroethane, etc.).
Specific examples of non-limiting non-aqueous liquids include 2-methyl-2-propanol, 2,3-dimethyl-2-butanol, 3-methyl-3-heptanol, 2-methyl-2-butanol (t-amyl alcohol), glycerin, N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), carbonates (e.g., dimethyl carbonate, ethyl methyl carbonate, diethyl carbonate, ethyl propyl carbonate, dipropyl carbonate, and isomers thereof), cyclic carbonates (e.g., ethylene carbonate, propylene carbonate, butylene carbonate, pentylene carbonate, and isomers thereof), hexamethylphosphoric triamide (HMPA), tetrahydrofuran (THF), N,N-dimethylacetamide, 2-pyrrolidone, N-methyl-2-pyrrolidone, tetramethyl urea, dioxane, dimethoxyethane, diethylene glycol dimethyl ether, ether, diisopropyl ether, 2-butanone (methyl ethyl ketone), 4-methyl-2-pentanone (isobutyl methyl ketone), 2-propanone, menthol, thymol, camphor, imidazole, coumarin, dimethylsulfone, urea, vanillin, camphene, salicylamide, pyridine, 2-aminopyridine, pyrimidine, piperidine, acetyl tributyl citrate, acetyl triethyl citrate, benzyl alcohol, butyrolactone, caprolactam, decylmethylsulfoxide, diacetin, diethyl phthalate, diethyl tartrate, dimethoxyethane, dimethylethylamide, 1-dodecylazacyclo-heptan-2-one, ethyl lactate, ethylene glycol, ethylene oxide, glycerol formal, glycofurol, propylene glycol, propylene oxide, silicone fluid, tetraglycol, triacetin, tributyl citrate, tributyrin, triethyl citrate, triethyl phosphate, the likes thereof, and combinations of two or more thereof.
Typically, a non-aqueous liquid having a relatively high water solubility, a relatively high solubility of non-volatile material in the liquid, a boiling point less than 100° C. and low toxicity is selected. Representative solvents include the foregoing and preferred solvents include tetrahydrofuran, tertiary alcohols such as 2-methyl-2-propanol, 2,3-dimethyl-2-butanol, 3-methyl-3-heptanol, 2-methyl-2-butanol (t-amyl alcohol), and ketones such as 2-butanone (methyl ethyl ketone), 4-methyl-2-pentanone (isobutyl methyl ketone), and 2-propanone. When the solubility of water in the liquid is sufficiently high, as with the foregoing solvents, the non-solvent can also be used to remove residual water from the composition, as described in more detail below. This advantage can be particularly useful when used in processing of multi-phasic dispersions. Surprisingly, substantially no disruption of the tertiary structure of any bioactive molecules occurs when the microspheres are contacted with or exposed to the non-solvent. 2-methyl-2-propanol has been found to be particularly useful in this regard and has been shown to facilitate lyophilization of excipients (e.g., sugars such as trehalose) leaving substantially only microparticles behind.
Certain non-aqueous liquids and their characteristics are listed in Table 1 below.













TABLE 1





Liquid
Densitya
BPb
FPc
Sol.d
LD50e
VPf





















2-butanone (MEK)
0.805
79.6
−86.3
25.6 
2.7
12


Acetone
0.786
56.2
−94.3
M
5.8
30.6


2-methyl-2-propanol
0.786
82.2
25.5
M
2.7
5.49


dimethoxyethane
0.868
83
−58
M
10
6.4


(ethylene glycol


dimethyl ether)


methyl t-butyl ether
0.741
55.2
−109
4.8
4
32.7


ether
0.713
34.6
−116.3
7.5
1.2
71.6


diisopropyl ether
0.724
68
−86
0.2
8.47
—





aDensity measured at ambient temperature such as 20-25° C.


bBoiling point (BP), ° C.


cFreezing/melting point (FP), ° C.


dSolubility (Sol) of water in the liquid in g/100 g, or ppm (parts per million by weight), at ambient temperature such as 20-25° C.


eLD50, in g/kg body weight, is the minimal amount of the liquid reported in the literature that when administered to rats orally all at once (acute toxicity) causes the death of 50% of the rats being tested.


fVapor pressure (VP), in kPa, at ambient temperature such as 20-25° C.


lMiscible (M) with water.






Exposing the Composition to the Non-Solvent and Removing the Non-Volatile Material

When the composition is combined with and/or otherwise exposed to the non-solvent, the physical and/or chemical characteristics of the non-solvent allow the at least one or more non-volatile materials in the composition to be solvated by the non-solvent while keeping the microparticles intact (as the microparticles are less soluble in the non-solvent than the non-volatile material). By removing the resulting liquid phases, the microparticles can be effectively separated from the one or more non-volatile materials and in some instances from one or more other components of the original composition. Non-limiting representative techniques useful to remove portions of the liquid phases and/or otherwise separate the microparticles from the composition or one or more components thereof include washing, filtration (including ultrafiltration), dialysis, diafiltration, phase separation (e.g., centrifugation), electrophoresis, and magnetic extraction, and combinations of two or more thereof. Centrifugation further includes ultracentrifugation, continuous flow centrifugation, and repeated centrifugal washing, and can be performed optionally in combination with liquid removal methods including but not limited to decantation and aspiration.
Certain components of the composition can be removed by selected techniques. For example, the one or more non-volatile materials of the composition can be substantially removed following one, two, or more repeated washings (e.g., by repeated centrifugal washing or repeated diafiltration) using the non-solvent, for example, typically at least 50%, such as 80%, 90%, 95%, 98%, 99%, or greater (e.g., 100%) of the non-volatile material can be removed. Additionally, water and/or other lyophilizable components in the composition, when present, can be removed partially or completely using lyophilization.
The process to separate the microparticles from the composition (or at least a portion of the non-volatile material) therein is typically carried out at a temperature above the freezing temperature of the composition or any component therein (or the continuous phase when a dispersion is being processed), and below the degradation temperature of the microparticles or the bioactive macromolecule therein, such as at or below ambient temperature, or above, at, or below temperatures such as 40° C., 37° C., 30° C., 25° C., 20° C., 15° C., 10° C., 5° C., 2° C, 0° C., −5° C., −10° C., −15° C., −20° C., or in a range between any two of such temperatures.
In one example, at a temperature at or below ambient temperature (such as 2-8° C.), the composition is a dispersion and is subjected to a diafiltration-based concentration process using a non-solvent as the diafiltration medium to exchange with and thereby remove at least a portion of the one or more components of the continuous phase (including the non-volatile material(s) and/or the solvent). Accordingly, the volume of the continuous phase may be reduced to concentrate the microparticles before drying (such as by lyophilization or air drying ) is carried out, thereby reducing processing time and cost. A diafilatration apparatus containing a peristaltic pump, a reservoir vessel, a hollow fiber cartridge, and tubing, as known to one of ordinary skill in the art, may be used for the concentration process. Centrifugal washing can be used similarly.
As a non-limiting result of removing one or more components of the continuous phase, an intermediate dispersion containing the microparticles and the non-solvent may be formed through the concentration process, wherein the concentration of the microparticles therein may be elevated many fold (such as 2 fold or more, 5 fold or more, 10 fold or more, 20 fold or more, or 40 fold or more) relative to that of the original dispersion, to 1 g/mL or greater, such as 10 mg/mL or greater. The one or more non-volatile materials (e.g., nonionic polymers) in the original continuous phase may be partially or completely removed during this concentration process. Thus, the continuous phase of the new dispersion may be different from that of the original dispersion in that it is at least substantially free of the one or more non-volatile materials therein. For example, the continuous phase of the new dispersion can contain less than 5% by weight or volume of the one or more non-volatile materials therein, such as less than, or equal to values such as 3%, 2%, 1%, 0.5%, or be present in a range between any two of such values after conducting one or more of the foregoing steps. The microparticles can be suspended freely in the new dispersion, or can be in the form of one or more re-suspendable aggregates (such as isopycnic bands or solid pellets).
The concentrated intermediate dispersion may then be processed using techniques such as dilution and/or repeated centrifugal washing using the same non-solvent that forms the intermediate dispersion or a different non-solvent (such as those disclosed herein) to further remove the one or more non-volatile materials (e.g., nonionic polymers), if any remain present. The separation process may result in a new dispersion containing the microparticles and the non-solvent used during the separation process, with a concentration of the microparticles in a range of 1 mg/mL to 50 g/mL, such as 5 g/mL to 20 g/mL, for example, about 10 g/mL. This new suspension may be stored and/or used as is, or optionally further processed to remove the non-solvent and yield the microparticles in a dry powder form. Non-limiting techniques to remove the non-solvent include various drying techniques (e.g., air drying, lyophilization, freeze-drying, liquid-phase drying, spray drying such as cold spray drying, cryogenic drying, spray-freeze drying, supercritical drying such as supercritical fluid drying, fluidized bed drying), and combinations of two or more thereof.
Alternatively, or in conjunction with one of the foregoing drying techniques, a humidified gas such as humidified nitrogen, humidified air, or humidified noble gases, can optionally be directed or passed over the obtained microparticles to beneficially exchange a portion of any remaining residual solvent therein with water and/or effectively dry the particles. Specifically, the microparticles are placed in a supporting member (e.g., vials, trays, pans, dishes), which is in turn placed in a chamber (e.g., dry box, lyophilizer, desiccator) under a stream of the humidified gas. Contacting the microparticles with a humidified gas having a relative humidity of 25% to 100%, such as 30% to 95%, 40% to 90%, or 50% to 80%, facilitates the evaporation of residual solvent from the microparticles. Following the high humidity evaporation process, which may last from hours (e.g., 10, 12, 20 hrs) to days depending on the volume or weight of the microparticle composition, the residual solvent content therein can be further reduced. If the residual solvent is not water miscible, a gas containing little to no humidity can be used to achieve similar benefits.
In one example, a flowable dispersion (e.g., a suspension) containing a plurality of microparticles, such as solid microspheres, is dispersed in a continuous phase containing water or an aqueous solution (such as a buffer solution, optionally with one or more polyvalent cations) with one or more non-volatile materials (e.g., nonionic polymers) solubilized therein. Optionally, the dispersion may be frozen at a temperature of −20° C. or less, preferably −40° C. or less, such as about −60° C., and lyophilized over a sufficient period of time (such as one day or longer, preferably 3 days) to remove substantially all of the water and any other lyophilizable substances to provide a solid dispersion (e.g., lyophilized cake) in which the microparticles may be dispersed in a solid continuous phase of the one or more non-volatile materials. Both the original flowable dispersion and the solid dispersion may be exposed to one or more non-solvents of the present disclosure such as by combining the microparticles with the non-solvent followed with agitation such that the microparticles may be dispersed in a single liquid phase containing the non-solvent (optionally, further containing a portion or all of the continuous phase of the original flowable dispersion). The one or more non-volatile materials may be dissolved or otherwise solubilized in the non-solvent while the microparticles remain dispersed as a solid phase. The new dispersion may be subjected to centrifugation with the supernatant aspirated or decanted away, thereby removing at least a portion of the one or more non-volatile materials solubilized therein from the microparticles. The microparticles may optionally be centrifugally washed more than once with the same non-solvent or with one or more different non-solvents when desired. Any residual non-solvents in the retained microparticles may be removed by drying the microparticles using one or more drying means known to one of ordinary skill in the art (e.g., under nitrogen stream or vacuum).
As previously described, the physical and/or chemical properties of the one or more non-solvents may be chosen such that it is relatively simple, convenient, and/or cost-effective to remove the remainder of the one or more non-solvents from the microparticle (e.g., through lyophilization, diafiltration, air-drying, or combinations thereof) to yield, for example, a dry powder of the microparticles. Alternatively or in addition, the non-solvent may be suitable as a carrier for the intended storage and/or end uses of the microparticles, thus rendering removal unnecessary or only partial removal desirable.
Continuous Phase The continuous phase of a multi-phasic dispersion processed in accordance with the disclosed methods may be non-solid, for example, containing one fluid or a mixture of two or more fluids (e.g., a homogeneous mixture of two or more liquids wherein at least a first liquid may be soluble in or miscible with at least a second liquid). Non-limiting examples of suitable fluids include aqueous fluids (e.g., water H2O, D2O, aqueous buffers, and other aqueous solutions), non-aqueous fluids (e.g., organic fluids, organic buffers), and combinations of two or more of the foregoing. In one aspect, the non-solid continuous phase may be substantially aqueous, for example, containing more than 10% by volume, such as 25% or more, 50% or more, or 75%, or more water. The continuous phase may be partially or completely aqueous or aqueous-miscible, aqueous-immiscible, water-soluble, or water-insoluble.
When measured at the same ambient temperature, such as 20° C. or 25° C., the continuous phase or the at least one liquid therein typically have density similar or equal to, or less than that of the dispersed phase or the microparticles therein. Most typically, the continuous phase or the at least one liquid therein have a density less than that of the microparticles. For example, the continuous phase or the at least one liquid therein may have a density at ambient temperature that is less than or equal to values such as 1.10 g/cm3, 1.05 g/cm3, 1.0 g/cm3, 0.95 g/cm3, 0.9 g/cm3, 0.8 g/cm3, 0.7 g/cm3, 0.6 g/cm3, or in a range between any two of such values.
The continuous phase may further contain one or more ingredients solubilized therein which are often not substantially incorporated into the microparticles including but not limited to the non-volatile material(s), salts, ions, excess reagents, excipients (e.g., sugars, polyols, surfactants), and/or manufacturing-related compounds. It should be noted, however, that the non-volatile material may be present on/in the dispersed phase, too, for example, the non-volatile material may be trapped within pores of the microparticles and/or otherwise associated with the microparticles. Additionally, these ingredients can be used in the non-solvent described herein. Non-limiting examples of salts include ammonium acetate, ammonium bicarbonate, and other buffer salts known to one of ordinary skill in the art. Non-limiting examples of sugars include trehalose, sucrose, lactose, and other carbohydrates known to one of ordinary skill in the art. Non-limiting examples of polyols include mannitol and other sugar alcohols known to one of ordinary skill in the art. The one or more fluids and/or solutes of the continuous phase may be, independently, partially or fully aqueous-miscible, aqueous-immiscible, water-soluble, and/or water-insoluble.
Dispersed Phase
The dispersed phase of a multi-phasic dispersion processed in accordance with the disclosed methods may comprise solid microparticles. Typically, it is preferred that the microparticles are substantially insoluble in and/or substantially immiscible with the non-solvent, or at least the non-aqueous liquid therein, for example, having a solubility therein at ambient temperature of less than 10% by weight, such as 5 wt. % or less, 3 wt. % or less, I wt. % or less, 0.5 wt. % or less, 0.1 wt. % or less, 0.05 wt. % or less, 0.01 wt. % or less, or in a range between any two of such values.
The dispersed phase may further include other materials in association with the solid microparticles, for example, a non-volatile material, salt, or excipient added during microparticle formation. Generally, such materials are not desired in the isolated microparticles and are therefore desirably removed from the dispersion. Accordingly, it is desirable for such materials to have relatively higher solubilities in the non-solvent described above.
The following examples are provided to illustrate the invention, but are not intended to limit the scope thereof.
EXAMPLE 1
Recombinant α-1-anti trypsin (rAAT) microspheres were made by cooling a solution containing 16% (w/v) PEG 3350, 0.02% (w/v) Pluronic® F-68, 10 mM ammonium acetate, and 2 mg/mL rAAT at pH 5.9 to 2° C., in accordance with the methods disclosed in U.S. Patent Application Publication No. 20050142206, which is incorporated herein by reference in its entirety. The resulting suspension containing rAAT microspheres (average particle size about 1-3 microns in diameter) was thoroughly mixed with a volumetric excess (10 ml, 20 ml, or 40 ml) of a polar aprotic liquid (acetone) at about 2-4° C. or about −20° C. It was unexpectedly found that PEG is insoluble in anhydrous acetone but very soluble in aqueous acetone (having a water content of greater than 4% by volume, such as 4.5% or 4.8% or more). The aqueous acetone suspensions were centrifuged at 2-4° C. and 2,000 rpm for 5-10 minutes, and the supernatants were removed. The remaining microspheres were washed twice more with the same liquid (aqueous acetone), and then dried under a stream of N2 gas. The percentage content of the microparticles that retained a spherical shape after being contacted with aqueous acetone was assessed using light microscopy. The percentage yield of the active agent in the microparticles was assayed using Bicinchoninic Acid (BCA) assay. The results are listed in Table 2 below. At temperatures greater than −20° C. (such as 2-4° C.) and a liquid to suspension volume ratio of 40:1 or less, the acetone washing process provided the best combination of high microparticle morphology retention and high specific activity (less denaturation).












TABLE 2







Wash
Liquid to
%




Temperature
Suspension
Microparticles



(° C.)
Volume Ratio
being Spherical
% Yield









−20
10:1
10%
70%



2-4
10:1
90%
76%



2-4
20:1
90%
91%



−20
40:1
10%
75%



2-4
40:1
90%
78%










EXAMPLE 2
A first solution (2.4 mg/ml IgG in 100 mM ammonium acetate buffer at pH 5.8) and a second solution (24 weight percent poloxamer 188 in 100 mM ammonium acetate buffer at pH 5.8) were pre-heated to 48° C. and mixed at a volume ratio of 1:1. The transparent solution (1.2 mg/ml IgG, 12% poloxamer 188, 100 mM ammonium acetate, pH 5.8) was equilibrated at 48° C. and then cooled to about 8° C. (but not frozen) to form a 1 mg/ml IgG microsphere suspension. The suspension was equilibrated (not frozen) in a 2-8° C. cold room, and then concentrated 40-fold using diafiltration. The concentrated suspension (40 mg/ml IgG microsphere) was then diluted to about 10 mg/ml with an aqueous 2-methyl-2-propanol solution (60% v/v 2-methyl-2-propanol, 40% v/v deionized water) using diafiltration. The diluted suspension was centrifuged, and the supernatant was removed. The resulting microspheres were centrifugally washed with the 60% aqueous 2-methyl-2-propanol solution five times, flash-frozen, and then lyophilized using a lyophilization cycle which included a warming phase at 30° C. to form a dry IgG microsphere powder substantially free of excipients.
EXAMPLE 3
A first solution (2.5 mg/ml IgG in 100 mM ammonium acetate buffer at pH 5.8) and a second solution (25 weight % PEG 8000 in 100 mM ammonium acetate buffer at pH 5.8) were pre-heated (warm water bath) to 50° C. and 55° C., respectively. The second solution was added to the first solution at a volume ratio of 1:1 and the mixture was gently mixed (using inversion). The transparent solution (1.25 mg/ml IgG, 12.5% PEG 8000, 100 mM ammonium acetate) was cooled to about 6° C. (but not frozen) to form 100 ml of a 1 mg/ml IgG microsphere suspension. The suspension was equilibrated (not frozen) in a 2-8° C. cold room, and then centrifuged to discard the supernatant. The remaining pellet was re-suspended in an aqueous 2-methyl-2-propanol solution (60% v/v 2-methyl-2-propanol, 40% v/v deionized water). The re-suspension was flash-frozen and then lyophilized to form a dry IgG microsphere powder.
EXAMPLE 4
A first solution (2.3mg/ml IgG in 100 mM ammonium acetate buffer at pH 5.8) and a second solution (24 weight % poloxamer 188 in 100 mM ammonium acetate buffer at pH 5.8) were pre-heated (warm water bath) to 50° C. The second solution was added to the first solution at a volume ratio of 1:1 and gently mixed (using inversion). The transparent solution (1.15 mg/ml IgG, 12% poloxamer 188, 100 mM ammonium acetate, pH 5.8) was equilibrated (warm water bath) at 50° C. and then cooled to about 6° C. (but not frozen) to form 1000 ml of a 1 mg/ml IgG microsphere suspension. As in Example 2, the suspension was concentrated and buffer exchanged using the 60% aqueous 2-methyl-2-propanol solution, flash-frozen, and then lyophilized to form a dry 1 gG microsphere powder.
EXAMPLE 5
A first solution (2.3 mg/ml IgG in 100 mM ammonium acetate buffer at pH 5.8) and a second solution (24 weight % poloxamer 188 in 100 mM ammonium acetate buffer at pH 5.8) were pre-heated (warm water bath) to 50° C. The second solution was added to the first solution at a volume ratio of 1:1 and gently mixed (using inversion). The transparent solution (1.15 mg/ml IgG, 12% poloxamer 188, 100 mM ammonium acetate, pH 5.8) was equilibrated (warm water bath) at 50° C. and then cooled to about 6° C. (in a −20° C. freezer for about 40 minutes, but not frozen) to form 2000 ml of a 1 mg/ml IgG microsphere suspension. The suspension was concentrated and buffer exchanged as described in Example 2, except that the 60% aqueous 2-methyl-2-propanol solution further contained 10 mM histidine and 10 mM ammonium acetate. Following flash-freezing and lyophilization, a dry IgG microsphere powder was formed.
EXAMPLE 6
Nucleic acid microparticles were formed according to the disclosure of U.S. Provisional Patent Application No. 60/938,123, the entirety of which is incorporated herein by express reference thereto. Following the formation of the microparticles, the suspensions were centrifuged, the supernatants decanted, and the pellets of microparticles retained. The pellets of microparticles were each re-suspended at 4° C. in 1 ml of an aqueous 60% 2-methyl-2-propanol solution (60% v/v 2-methyl-2-propanol, 40% v/v deionized nuclease-free water), and centrifugally washed (centrifuged, the supernatants decanted, and the pellets of microparticles retained). The centrifugal washing was repeated two more times. The pellets of microparticles were each re-suspended in 0.5 ml of the aqueous 60% 2-methyl-2-propanol solution, and placed at −80° C. to freeze the re-suspension. The frozen samples were lyophilized to provide dry powders of the respective nucleic acid microparticles.
EXAMPLE 7
IgIV microparticles were formed according to established methods for forming microparticles comprising antibodies, for example, as disclosed in Example 2. The microsphere suspension was lyophilized in 10 ml glass vials with fills of 2 ml. The employed lyophilization cycle included freezing the suspensions, annealing the suspensions, ramping the temperature to conduct the primary drying step, conducting the primary drying step, and then optionally performing a secondary drying step (5A and/or 5B), as shown in Table 3. Backfilling with air at was conducted 20° C. when the cycle was completed.












TABLE 2







Shelf






Ramp
Temperature
Vacuum
Hold Time


Step
(° C./min)
(° C.)
(mT)
(min)
Description




















1
2.5
−40
N/A
120
Freezing


2
1.0
−20
N/A
240
Annealing


3
2.5
−30
N/A
30
Ramp to







primary


4
N/A
−30
100
1200
Primary drying


5A
0.5
−20
100
0
Secondary







Drying


5B
0.5
20
100
0
Drying at 20° C.









The chemical integrity of the IgIV microparticles was determined after lyophilization using HPLC assays (Size Exclusion Chromatography (SEC)) to quantitate agglomeration and degradation products. Results demonstrated that there was no significant accumulation of agglomerates or other related substances during the lyophilization process.
Approximately half of the samples were placed inside a desiccator connected to a humidified nitrogen apparatus. The humidified nitrogen was at 50% relative humidity for approximately 12 hours (20 psi), followed by 6 hours at 0% relative humidity (20 psi). Results demonstrated that a dried powder containing less than 1.0 wt. % insoluble antibody, less than 0.5 wt. % residual non-volatile material was obtained. Results further demonstrated a significant reduction in residual (non-)solvent content (12% decrease in 2-methyl-2-propanol content) although a slight increase in residual moisture (2% increase) and high molecular weight species (1.3% increase) was observed relative to the dried powder immediately after lyophilization. Nonetheless, these results demonstrate successful removal of the non-volatile material from the microparticles, and the ability to dry the powder while maintaining protein integrity.

## Claims

1. A method of separating a non-volatile material from microparticles comprising:
providing a composition comprising a plurality of solid microparticles and at least one non-volatile material,
providing a non-solvent comprising at least one non-aqueous liquid selected from the group consisting of tetrahydrofuran, 2-methyl-2-propanol, 2,3-dimethyl-2-butanol, 3-methyl-3-heptanol, 2-methyl-2-butanol, 2-butanone, 4-methyl-2-pentanone, 2-propanone, and combinations thereof and at least one aqueous liquid;
exposing the composition to the non-solvent to form a mixture containing one or more liquid phases and the solid microparticles; and
removing at least a portion of the resulting one or more liquid phases while retaining at least the microparticles, thereby removing at least a portion of the non-volatile material from the composition,
wherein the non-volatile material is more soluble in the non-solvent than are the microparticles, and
the non-solvent is free of primary, linear alcohols and halogenated solvents.

2. The method of claim 1, wherein the microparticles are soluble in the aqueous liquid, but have less than 5 wt. % solubility in the non-solvent.

3. The method of claim 1, wherein the non-aqueous liquid is soluble in or miscible with the aqueous liquid.

4. The method of claim 1, wherein the solid microparticles are substantially insoluble in the at least one non-aqueous liquid.

5. The method of claim 1, wherein the exposing and/or removing steps comprise washing, diafiltration, filtration, dialysis, electrophoresis, or a combination thereof.

6. The method of claim 1, further comprising drying the microparticles to form a powder.

7. The method of claim 1, wherein the non-solvent has an oral LD50 in rats of greater than or equal to 1.6 g/kg by body weight.

8. The method of claim 1, wherein the non-solvent has a density less than or equal to 0.95 g/ml.

9. The method of claim 1, wherein the at least one non-volatile material is water-soluble.

10. The method of claim 1, wherein the non-solvent comprises 1% to 95% by volume of the at least one aqueous liquid.

11. The method of claim 1, wherein the non-solvent comprises water and a second fluid component selected from the group consisting of 2-methyl-2-butanol (t-amyl alcohol), glycerin, N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), acetone, carbonates, cyclic carbonates, hexamethylphosphoric triamide (HMPA), tetrahydrofuran (THF), N,N-dimethylacetamide, 2-pyrrolidone, N-methyl-2-pyrrolidone, tetramethyl urea, dioxane, dimethoxyethane, diethylene glycol dimethyl ether, ether, diisopropyl ether, 2-butanone (methyl ethyl ketone), 4-methyl-2-pentanone (isobutyl methyl ketone), 2-propanone, menthol, thymol, camphor, imidazole, coumarin, dimethylsulfone, urea, vanillin, camphene, salicylamide, pyridine, 2-aminopyridine, pyrimidine, piperidine, acetyl tributyl citrate, acetyl triethyl citrate, benzyl alcohol, butyrolactone, caprolactam, decylmethylsulfoxide, diacetin, diethyl phthalate, diethyl tartrate, dimethoxyethane, dimethylethylamide, 1-dodecylazacyclo-heptan-2-one, ethyl lactate, ethylene glycol, ethylene oxide, glycerol formal, glycofurol, propylene glycol, propylene oxide, silicone fluid, tetraglycol, triacetin, tributyl citrate, tributyrin, triethylcitrate, triethyl phosphate, and combinations thereof.

12. The method of claim 1, wherein the non-solvent comprises at least 40% by volume of 2-methyl-2-propanol.

13. The method of claim 1, wherein the solid microparticles comprise an active agent.

14. The method of claim 1, wherein the solid microparticles comprise at least one bioactive macromolecule on an outer surface of the solid microparticle.

15. The method of claim 14, wherein the at least one bioactive macromolecule is selected from the group consisting of carbohydrates, peptides, proteins, vectors, nucleic acids, complexes thereof, conjugates thereof, and combinations thereof.

16. The method of claim 1, wherein the solid microparticles comprise a carrier macromolecule.

17. The method of claim 1, wherein the at least one non-volatile material is selected from the group consisting of nonionic polyethers, nonionic copolyethers, nonionic polyesters, nonionic copolyesters, nonionic polyether-polyester copolymers, non-ionic vinyl polymers, non-ionic pyrrolidone-containing polymers, non-ionic polymeric carbohydrates, derivatives and salts of the foregoing materials, and combinations thereof.

18. The method of claim 1, wherein the microparticles are amorphous, spherical, or both.

19. The method of claim 1 wherein the non-solvent is a single-phase mixture comprising at least two liquids.

20. The method of claim 1, further comprising isolating the microparticles.

21. The method according to claim 20, further comprising lyophilizing the isolated microparticles.

22. The method according to claim 20, further comprising passing humidified nitrogen over the isolated microparticles.

23. The method of claim 1, wherein the composition is a multi-phasic dispersion comprising dispersed and continuous phases, the dispersion comprising the solid microparticles, the at least one non-volatile material, and a solvent.

24. The method of claim 23, wherein the solvent is soluble in or miscible with the non-solvent.

25. A method of separating a non-volatile material from microparticles comprising:
providing a composition comprising a plurality of solid microparticles and at least one non-volatile material,
providing a non-solvent comprising 2-methyl-2-propanol;
exposing the composition to the non-solvent to form a mixture containing one or more liquid phases and the solid microparticles; and
removing at least a portion of the resulting one or more liquid phases while retaining at least the microparticles, thereby removing at least a portion of the non-volatile material from the composition,
wherein the non-volatile material is more soluble in the non-solvent than are the microparticles.

26. The method of claim 25, wherein the non-solvent further comprises water.

27. The method of claim 25, wherein the microparticles have less than 5 wt. % solubility in the non-solvent.

28. The method of claim 27, wherein the solid microparticles are substantially insoluble in 2-methyl-2-propanol.

29. The method according to claim 27, wherein the composition is a multi-phasic dispersion comprising dispersed and continuous phases, the dispersion comprising the solid microparticles and at least one of a non-volatile material and a solvent.

30. The method of claim 29, wherein the solvent is soluble in or miscible with the non-solvent.

31. A method of separating a non-volatile material from microparticles comprising:
providing a composition comprising a plurality of solid microparticles, water, and at least one non-volatile material,
providing a non-solvent comprising at least one non-aqueous liquid and at least one aqueous liquid;
exposing the composition to the non-solvent to form a mixture containing one or more liquid phases and the solid microparticles; and
removing at least a portion of the resulting one or more liquid phases while retaining at least the microparticles, thereby removing at least a portion of the non-volatile material and at least a portion of the water from the composition,
wherein the non-volatile material is more soluble in the non-solvent than are the microparticles, and
the at least one non-aqueous liquid is selected from the group consisting of tetrahydrofuran, 2-methyl-2-propanol, 2,3-dimethyl-2-butanol, 3-methyl-3-heptanol, 2-methyl-2-butanol, 2-butanone, 4-methyl-2-pentanone, 2-propanone, and combinations thereof.

32. The method of claim 31, wherein the microparticles have less than 5 wt. % solubility in the non-solvent.

33. The method of claim 31, wherein the solid microparticles are substantially insoluble in the at least one non-aqueous liquid.

34. The method according to claim 31, wherein the composition is a multi-phasic dispersion comprising dispersed and continuous phases, the dispersion comprising the solid microparticles and at least one of a non-volatile material and a solvent.

35. The method of claim 34, wherein the solvent is soluble in or miscible with the non-solvent.

36. The method of claim 1, wherein the microparticles comprise a bioactive macromolecule having a tertiary structure.