Compositions and methods for reducing adverse effects of storage, transport and administration of antigen-containing formulations - Patents.com

Abstract

Embodiments of the present disclosure generally relate to novel compositions, methods, and systems for reducing or eliminating the effects of interfering agents or premature dissolution of antigen-containing microparticles during storage, transportation, and delivery of antigen-containing formulations. Particular embodiments relate to improved formulations for more reliable storage, transportation, delivery, and administration of metal oxide coated antigen-containing suspension formulations.

Description

[Technical field]

[0001] Embodiments of the present disclosure generally relate to novel compositions, methods, and systems for reducing or eliminating the effects of interfering agents or premature dissolution of antigen-containing complexes during storage, transportation, and delivery of antigen-containing formulations. Certain embodiments relate to improved and reliable storage, transportation, and delivery of formulations, including antigen-containing suspensions or suspension formulations coated with metal oxides. [Background technology]

[0002] There are several challenges presented by storing liquid formulations of antigen-containing drugs in vials and syringes. Some formulations, including therapeutic proteins, are adversely affected when exposed to hydrophobic interfaces (e.g., interfaces presented by silicone oil lubricants, elastomeric stoppers, and septa). Container-liquid interfaces, such as air-water interfaces and water-container interfaces, are other sources of potential degradation of therapeutic products, or adverse effects, that can destabilize antigen-containing formulations during storage and reduce product delivery.

[0003] Other problems include the instability of the target agent in the liquid formulation, which may cause the target agent to be destabilized or undesiredly coupled due to adverse molecular interactions with antimicrobial agents and preservatives. Using dry formulations, which alleviates some of the problems with liquid formulations but creates other problems, creates other problems. For example, filling vials with dry powders is more difficult than filling liquid formulations, and dry powders and lyophilized formulations typically must be reconstituted with water or buffer before administration. Furthermore, when preservatives and antimicrobial agents are added to the reconstitution medium, the stability of the antigen-containing product may be compromised due to interference between the preservatives and antimicrobial agents and the target agent. Thus, there is a need to improve the ease and more accurate delivery of storage, transportation, and delivery of antigen-containing agents that are susceptible to interference, irregularities in distribution, and degradation due in part to these specific factors. Summary of the Invention [Means for solving the problem]

[0004] The embodiments of the present disclosure generally relate to novel compositions, methods and systems for improving the stability of time-release formulated antigens, the stability of coated antigens, and reducing or eliminating the effect of interfering agents on the storage, transportation and delivery of antigen-containing formulations. Some embodiments relate to stabilizing metal oxide coated antigens in the form of suspended particles or microparticles, whereby the suspension formulations disclosed herein maintain the continuity and stability of the coated antigen particles. Certain embodiments relate to improved reliable storage, transportation and delivery of coated or layered antigen-containing formulations to improve the reliability of delivery of antigens used to improve, treat or prevent health conditions in subjects.

[0005] In some embodiments, the coated or layered antigens disclosed herein include, but are not limited to, metal oxide coated antigens. According to these embodiments, the metal oxide coated antigens may include coated spray freeze dried antigens in a glassy state. In certain embodiments, the coated antigens may include atomic layer deposition (ALD) coated antigens (e.g., antigenic proteins, polypeptides, polysaccharides, polynucleotides, microorganisms (e.g., viruses, fungi, bacteria, prions), small molecules, or fragments thereof) that have undergone a drying process before being coated. According to certain embodiments, the ALD coated antigens may be in the form of particles, such as microparticles in a dry or powder state, to improve the stability, drug compatibility, storage, and transportation of the coated antigen(s). In certain embodiments, the metal oxide coated (e.g., ALD coated) microparticles containing one or more antigens may be suspended for delivery to a subject, and the suspension buffer includes a non-chelating organic drug alone or in combination with other drugs.

[0006] In some embodiments, the formulations disclosed herein can include combinations of one or more ALD coated microparticles containing the same or different antigens, and can further include, but are not limited to, formulations to improve microparticle integrity and reduce storage component interference and additive interference for improved and reliable delivery of coated antigens to a subject. The embodiments disclosed herein provide formulations for long-term storage and immediate administration of metal oxide coated antigen-containing microparticles for efficient and precise delivery of targeted antigens to prevent, treat, or reduce the onset of a condition or infection.

[0007] In some embodiments, the present disclosure relates to improved storage and delivery of previously disclosed controlled release powdered vaccine formulations (see, e.g., PCT / US2017 / 019163, filed Feb. 23, 2017). In certain embodiments, a combination of spray drying and atomic layer deposition (ALD) can be used to coat antigen particles embedded in a glassy matrix for improved stabilization. In certain embodiments, the glassy matrix can be provided by any agent known in the art that can form a glassy matrix while in an essentially dry form (e.g., trehalose, sucrose, mannitol, and sucrose or similar).

[0008] In other embodiments, the target antigens or antigens contemplated herein include, but are not limited to, immunogenic agents, such as polypeptides, polynucleotides including RNA, DNA, hybrid molecules, lipid-coated polynucleotides, virus-like particles, viruses, bacteria-derived agents, bacteriophages or agents derived therefrom, polysaccharides, chimeric polypeptides or polynucleotides, or combination polynucleotides, toxins, microorganisms or fragments thereof, small molecules, or fragments or derivatives thereof, and can be formulated into coating-ready particles, such as glassy matrices, that are stable. In accordance with these embodiments, the essentially dry microparticles of coatable antigens can include formulations known in the art for use in preparing coatable particles, including, but not limited to, trehalose, sucrose, mannitol, and sucrose or similar agents or combinations thereof. (See, e.g., PCT / US2015 / 029529, filed May 6, 2015, which is incorporated herein by reference in its entirety).

[0009] In other embodiments, the antigens contemplated herein for use in treating, reducing the onset of, or preventing a health condition may be readily available for metal oxide or other suitable metal ion coating. It is contemplated herein that any metal oxide or metal ion coated antigen-containing microparticles may be in a dry or essentially dry state that can be prepared for suspension in a non-chelating organic buffer with reduced or low ionic strength as contemplated herein for long term storage at ready-to-use conditions.

[0010] In some embodiments disclosed herein, powders produced by the processes disclosed herein (e.g., ALD-coated microparticles containing antigens) can be suspended or resuspended in an aqueous medium with reduced interference from storage components and / or agents used to preserve the composition. According to these embodiments, the aqueous medium includes, but is not limited to, a non-ionic tonicity modifier-containing buffer. In some embodiments, the buffer includes a non-chelating organic buffer. In certain embodiments, the non-chelating organic buffer includes, but is not limited to, histidine or similar non-chelating organic buffer. In other embodiments, the buffer or aqueous medium cannot be an inorganic buffer capable of dissolving the metal oxide coating, or one that forms a chelate with, for example, a phosphate or citrate buffer, or a phosphate-citrate combination buffer, a solution containing a chelating agent (e.g., ethylenediaminetetraacetic acid (EDTA)), or other agent that can dissolve or reduce the integrity of the metal oxide coating. In certain embodiments, the formulation for the coated microparticles disclosed herein can include histidine and / or phosphate (e.g., sodium phosphate). According to these embodiments, the concentration of histidine and / or phosphate can be about 40 mM or less; or about 30 mM or less; about 20 mM or less; about 10 mM or less; or about 5 mM or less. In some embodiments, the pH of the suspension formulation containing histidine and / or phosphate is less than about pH 8.0 when the concentration of histidine and / or phosphate is about 40.0 mM or less, 30 mM or less, 20 mM or less, or 10 mM or less. In some embodiments, the pH of the suspension formulation containing histidine and / or phosphate is less than about pH 8.0 when the concentration of histidine and / or phosphate is about 5.0 mM or less. In some embodiments, the pH of the suspension formulation containing histidine and / or phosphate is about pH 7.0 or more when the concentration of histidine and / or phosphate is about 5.0 mM or less.In certain embodiments, the pH of the histidine-containing suspension formulation is about 7.0 or greater, or about 7.0 to about 8.5, or about 7.5 to about 8.5 when the concentration of histidine is less than 20 mM; or 15 mM or less; or 10 mM or less; or 5 mM or less. In certain embodiments, these suspension formulations can further comprise one or more additional agents to stabilize and / or facilitate delivery of the coated microparticles containing one or more antigens.

[0011] In some embodiments, the suspension formulation for the coated antigen-containing microparticles may contain histidine and may be stored at any temperature for storage, transportation, and delivery to a subject. According to these embodiments, the suspension formulation containing histidine may be stored at temperatures below about 4°C to above about 60°C, or above about 50°C, for a period of several hours to about one week, one month, two months, or more without premature or unscheduled release of one or more coated antigens. In some embodiments, these suspension formulations do not contain phosphate. In certain embodiments, these formulations may further include one or more additional agents (e.g., trehalose) to stabilize and / or facilitate delivery of the coated microparticles containing one or more antigens. In some embodiments, the suspension formulation for the coated antigen-containing microparticles can include histidine and trehalose (or sucrose or similar agent) and can be stored at temperatures below about 4° C. to above about 60° C., or above about 50° C., for a period of several hours to about a week, a month, two months or more without premature or unscheduled release of one or more coated antigens, enhancing storage, transport and delivery of the coated antigen-containing microparticles. In other embodiments, the suspension formulations disclosed herein do not include aqueous phosphate, citrate, or sulfate. In other embodiments, the suspension formulations disclosed herein do not include a chelating agent. In other embodiments, the suspension formulations disclosed herein do not include EDTA. In other embodiments, the suspension formulations disclosed herein can further include a salt at a concentration of less than 0.1 M.

[0012] In certain embodiments, the suspension formulations disclosed herein may further comprise trehalose or sucrose, etc. In certain embodiments, the concentration of trehalose and / or sucrose may be about 20% w / v or less. In certain embodiments, the concentration of trehalose and / or sucrose may be about 15% w / v or less. In still other embodiments, the suspension formulations disclosed herein may comprise a surfactant, such as a polysorbate.

[0013] In some embodiments, the suspension formulation disclosed herein does not include a buffer containing citrate, phosphate, or its salt, such as sodium salt. In certain embodiments, the suspension formulation disclosed herein does not include a buffer containing citrate. In certain embodiments, the suspension formulation disclosed herein does not include a buffer containing citrate-phosphate formulation. According to these embodiments, citrate alone or in combination with phosphate induces instability of the coated antigen microparticles disclosed herein. In other embodiments, the suspension formulation disclosed herein does not include a buffer containing a phosphate buffer.

[0014] In other embodiments, the composition may include one or more surfactants or other similar agents. In other embodiments, the compositions disclosed herein may include an antibacterial agent. In yet other embodiments, the suspension or aqueous formulation used herein may include one or more preservatives (e.g., benzyl alcohol, methylparaben, creosol, phenol, etc.).

[0015] In other embodiments, with respect to the embodiments disclosed herein and at least paragraphs

[0006] to

[0015] above, the antigen or targeting agent in the suspended microparticles in a non-chelating buffer solution having one or more layers of a metal oxide coating (e.g., ALD) is thermally stable. Advantages of these formulations include, but are not limited to, providing a format in which the suspended particles can be more easily filled into vials, tubes, syringes, or other containers, for example, using conventional liquid fill-finish equipment, more efficiently than powders. Another advantage of using aqueous formulations is that the formulation as a stable suspension of powder or a stable suspension of metal oxide or ALD coated microparticles eliminates the need for suspension in preparation for administration of the vaccine, immunogenic composition, or therapeutic agent of interest, or at the time of administration. In addition, these stable formulations are ready-to-inject formulations of the suspended powder or microparticles, eliminating the need for a second vial of suspension, reducing shipping weight, and improving convenience for caregivers. In yet other embodiments, the metal oxide or ALD coated microparticles create a layer that protects antigens, such as polypeptides or polynucleotides or small molecules embedded within the ALD coated microparticle powder from damage by antimicrobials or other preservatives, extending the shelf life of single and multi-dose preservative-containing formulations. In yet other embodiments, antigens coated with atomic layer deposition (ALD) and suspended in an aqueous medium are protected or isolated from deleterious interactions with container surfaces, container closure elements (e.g., stoppers, septa, and syringe plungers), lubricants (e.g., silicone oils), and other interfaces.

[0016] Other embodiments disclosed herein relate to kits for the storage, transport and use of the suspended metal oxide coated antigens disclosed herein. In certain embodiments, the kits can include a ready-to-use syringe containing the suspension formulation disclosed herein for delivering a dose of the target antigen to a subject. Other embodiments relate to multi-dose formulations with improved stability and reliability.

[0017] The following drawings are intended to be illustrative and should not be construed as limiting in any way. [Brief description of the drawings]

[0018] [Figure 1] 1 shows the zeta potential of metal oxide coated microparticles in various buffers and pH according to certain embodiments disclosed herein. [Figure 2A] 2A and 2B depict exemplary graphs showing pre-mature leakage of antigen from coated particles in the presence and absence of various buffers, and in the presence or absence of surfactants, at room and elevated temperatures, with release into suspension buffer of certain embodiments disclosed herein (2A) by IR detection or SDS-PAGE, and (2B) by IR. [Figure 2B] 2A and 2B depict exemplary graphs showing premature leakage of antigen from coated particles in the presence and absence of various buffers, and in the presence or absence of surfactants, at room and elevated temperatures, with release into suspension buffer of certain embodiments disclosed herein (2A) by IR detection or SDS-PAGE, and (2B) by IR. [Diagram 3] 1 depicts plots demonstrating the release of antigen from coated particles analyzed by IR and SDS-PAGE of certain embodiments disclosed herein in the presence or absence of various buffers under different temperature conditions. [Figure 4] 1 depicts plots demonstrating the release of antigen from coated particles analyzed by IR and SDS-PAGE of certain embodiments disclosed herein in the presence or absence of various buffers under different temperature conditions over a time course of incubation. [Diagram 5]1 shows a plot of spray-dried microparticles of an exemplary antigen embedded in a glassy matrix coated with a metal oxide by atomic layer deposition (ALD). The coated microparticles are then suspended in an aqueous buffer and the release of the antigen from the microparticles into the buffer is measured in certain embodiments disclosed herein. [Figure 6] 1 shows a graph of the percent antigen release of coated microparticles suspended in a suspension buffer containing a chelating agent over several days of incubation in certain embodiments disclosed herein. [Figure 7] FIG. 1 depicts an illustrative schematic diagram of a metal oxide coat applied by ALD to a microparticle containing at least one antigen in certain embodiments disclosed herein. [Figure 8] FIG. 1 shows an exemplary process represented by a schematic showing metal oxide coating of an antigen in certain embodiments disclosed herein. [Figure 9A] 9A-9C show schematic diagrams of certain embodiments disclosed herein of an antigen coated by metal oxide layer formation (9A); a second, different antigen added to the outer layer of a partially coated particle (9B); and a second coating of the same or different antigen (9C). [Figure 9B] 9A-9C show schematic diagrams of certain embodiments disclosed herein of an antigen coated by metal oxide layer formation (9A); a second, different antigen added to the outer layer of a partially coated particle (9B); and a second coating of the same or different antigen (9C). [Figure 9C] 9A-9C show schematic diagrams of certain embodiments disclosed herein of an antigen coated by metal oxide layer formation (9A); a second, different antigen added to the outer layer of a partially coated particle (9B); and a second coating of the same or different antigen (9C). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] definition Unless specifically defined herein, a term has the meaning commonly understood by one of ordinary skill in the art as relating to or applicable to a particular embodiment disclosed herein.

[0020] Unless otherwise indicated, all numbers expressing properties such as amounts of agents and / or compounds, molecular weights, and reaction conditions disclosed herein are intended to be modified in all instances by the term "about". Thus, unless indicated to the contrary, the numerical parameters in the specification and claims are approximations that may vary by plus and / or minus about 10% to about 15%, depending on the desired properties sought as disclosed herein. Numerical values ​​expressed herein inherently include standard deviations necessarily resulting from errors found in numerical testing measurements.

[0021] As used herein, the term "subject" may refer to any mammal, including, but not limited to, a non-human primate (e.g., a monkey or ape), a farm animal or pet (e.g., a cow, pig, cat, dog, rat, mouse, horse, goat, rabbit, sheep, hamster, guinea pig) or other subject. In some embodiments, the mammalian subject is a human, such as an adult, child, adolescent, infant, baby or fetus.

[0022] Detailed Description In the following sections, specific exemplary compositions and methods are described to detail specific embodiments of the present invention.It is clear to those skilled in the art that implementing specific embodiments does not require the use of all or even some of the specific details outlined herein, but rather, concentrations, times and other specific details can be changed through routine experimentation.In some cases, well-known methods or components are not included in the description.

[0023] The embodiments of the present disclosure generally relate to novel compositions, methods, and systems for improving the suspension stability of time-release formulated antigens, the stability of the coated antigens, and reducing or eliminating the effect of interfering agents on the storage and delivery of antigen-containing formulations. Some embodiments relate to stabilizing metal oxide coated antigens in the form of dry particles once suspended, whereby the formulations disclosed herein maintain the stability of the coated antigen particles. Certain embodiments relate to improved reliable storage, transportation, and delivery of coated antigen-containing formulations.

[0024] In some embodiments, the coated antigens disclosed herein include, but are not limited to, metal oxide coated antigens, such as coated spray freeze dried antigens in a glassy state. According to these embodiments, the coated antigens may include atomic layer deposition (ALD) coated antigens (e.g., proteins, polypeptides, polynucleotides, microorganisms, small molecules, or fragments thereof) that have undergone a drying process or a thermal stability transition before being coated. According to certain embodiments, the ALD coated antigens may be in the form of particles, such as microparticles in an essentially dry or powdered state, to improve the stability, drug compatibility, storage, and transportation of the coated antigen(s). In certain embodiments, metal oxide coated (e.g., atomic layer deposition (ALD) coated) microparticles containing one or more antigens may be suspended for delivery to a subject, where the suspension buffer comprises a non-chelating organic agent of reduced ionic strength.

[0025] In some embodiments, the formulations disclosed herein can include combinations of one or more ALD coated microparticles containing the same or different antigens, and further include, but are not limited to, formulations to improve microparticle integrity (e.g., reduce or eliminate premature antigen release), reduce storage component interference, and reduce additive interference for improved and reliable antigen delivery to a subject.Embodiments disclosed herein provide formulations for long-term storage and ready administration of suspended metal oxide coated antigen-containing microparticles for efficient and precise delivery of targeted antigens to treat or reduce the onset of a pathology, or to treat or reduce the onset or spread of an infection.

[0026] In some embodiments, the present disclosure relates to improved storage and delivery of previously disclosed (see, e.g., PCT / US2017 / 019163, filed Feb. 23, 2017, which is incorporated herein by reference for all purposes) controlled release powder vaccine formulations. In these methods, a combination of spray drying and atomic layer deposition (ALD) can be used to coat antigen particles embedded in a glassy matrix. In other embodiments, target antigens (including but not limited to polypeptides, polynucleotides (including RNA, DNA), lipid-coated polynucleotides or combinations of polynucleotides, microorganisms, small molecules, virus-like particles, viruses, polysaccharides, or fragments or derivatives thereof) can be formulated into coating-ready particles (e.g., stable glassy matrices). According to these embodiments, the essentially dry microparticles of antigen ready for coating can include formulations known in the art in preparing particles ready for coating, including, but not limited to, trehalose, sucrose, or combinations thereof (see, e.g., PCT / US2015 / 029529, filed May 6, 2015, which is incorporated herein by reference in its entirety for all purposes). In some embodiments, the antigen-containing formulation can be lyophilized in the presence of a glass-forming excipient, and sufficient liquid can be removed during lyophilization such that the dried or essentially dry formulation or immunogenic composition exhibits a glass-stable state ready for coating with a metal oxide or other suitable metal ion. In other embodiments, the antigens contemplated herein for use in treating, reducing the onset of, or preventing a pathology can be readily available for metal oxide or other suitable metal ion coating. It is contemplated herein that any metal oxide or metal ion coated antigen-containing microparticle can be in a dry state ready for suspension in a non-chelating organic buffer contemplated herein for long-term storage in a ready-to-use state.

[0027] In some embodiments disclosed herein, powders (e.g., ALD-coated microparticles containing antigens) produced by the processes disclosed herein can be suspended or resuspended in an aqueous medium or suspension formulation that reduces interference or adverse effects from storage components and / or storage agents used to preserve the composition. According to these embodiments, the aqueous medium includes, but is not limited to, a non-ionic tonicity modifier-containing buffer. In some embodiments, the buffer comprises a non-chelating organic buffer. In certain embodiments, the non-chelating organic buffer or agent includes, but is not limited to, histidine, imidazole, glycine, bis-trismethane, tris, bicine, glycylglycine, or similar non-chelating organic buffers or other agents or buffers known to those skilled in the art as Good's buffers or amine buffers. In other embodiments, the buffers used in the suspension formulations disclosed herein include, but are not limited to, MES, bis-trismethane, ADA, bis-trispropane, PIPES, ACES, MOPSO, coramine chloride, MOPS, BES, TES, HEPES, DIPSO, MOBS, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, tricine, tris, glycinamide, glycylglycine, HEPBS, bicine, TAPS, AMPB, CHES, CAPSO, AMP, CAPS, CABS, etc. These buffers do not contain sulfate-containing agents. In other embodiments, the buffer or aqueous medium cannot contain any agent that can dissolve the metal oxide coating, such as phosphate or citrate buffers, or chelating phosphate-citrate combination buffers, or other agents that can dissolve or reduce the integrity of the metal oxide coating. In certain embodiments, phosphate, citrate, or similar chelating buffers are avoided or reduced in concentration to avoid dissolution of the metal oxide coating of the microparticles disclosed herein and to reduce pre-mature release of antigens from metal oxide coated particles or microparticles.

[0028] In other embodiments, the concentration of the organic buffering agent may be of reduced or minimal ionic strength, for example, to reduce interference (e.g., coating degradation or premature coating dissolution) that may occur with the coating of the target antigen or active agent coated with metal oxide. In certain embodiments, the organic buffering agent for the particle suspension contemplated herein reduces premature antigen release to improve the accuracy and predictability of dosing upon delivery to the subject. According to these embodiments, the inorganic or organic buffering agent may be about 100.0 mM or less, or about 50.0 mM or less, or about 25.0 mM or less, or about 10.0 mM or less, or about 5.0 mM or less, or about 1.0 mM or less. In other embodiments, the composition may include one or more surfactants (e.g., high molecular weight surfactants or other agents). In certain embodiments, the surfactant may include a non-ionic surfactant, including, but not limited to, polysorbate 80, polysorbate 20, or poloxamer 188 or poloxamer 403 or poloxamer 407, Tween 20®, Tween 80®, and the like. In other embodiments, the compositions disclosed herein may include an antimicrobial agent. In still other embodiments, the suspension or aqueous medium used herein may include one or more preservatives (e.g., benzyl alcohol, methylparaben, paraben, chlorobutanol, phenol, sorbic acid, cresol, meta-cresol, or other known preservatives). According to these embodiments, the resulting suspension is stable for about 1 day, about 1 week, about 2 weeks, several weeks, about 1 month, about 6 weeks, about 2 months, about 10 weeks, about 3 months, about 6 months, or more, while reducing or preventing release of antigen into the suspension medium.

[0029] In other embodiments, with respect to embodiments disclosed herein, the antigen or targeting agent in particles suspended in a non-chelating buffer solution having one or more layers of a metal oxide coating (e.g., ALD) is thermally stable at temperatures from below 4° C. to about 50° C. to about 55° C. or about 60° C. for about 1 day, about 1 week, about 2 weeks, several weeks, about 1 month, about 6 weeks, about 2 months, about 10 weeks, about 3 months, about 6 months or more.

[0030] Some advantages of the formulations disclosed herein include, but are not limited to, providing a format in which suspended particles can be filled more easily into vials, tubes, syringes, bottles, droppers, atomizers, or other containers than powders using conventional liquid fill-finish equipment. Another advantage is that formulation as a stable suspension of powder or stable suspension of metal oxide or ALD coated microparticles eliminates the need for suspension in preparation for or at the time of administration of the vaccine. In addition, these stable formulations are ready-to-inject formulations of suspended powders that eliminate the need for a second vial of suspension, reduce shipping weight, and improve convenience for caregivers. In yet other embodiments, the metal oxide or ALD coated microparticles create a layer that protects antigens, such as polypeptides or polynucleotides or small molecules embedded within the ALD coated microparticle powder from damage by antimicrobial agents or other preservatives, extending the shelf life of single and multi-dose preservative-containing formulations. In yet other embodiments, antigens coated with ALD and suspended in an aqueous medium are protected or isolated from adverse interactions with surfaces and other interfaces. According to these embodiments, the suspended ALD-coated microparticles protect the antigen (e.g., polypeptide, polynucleotide, microorganism, small molecule, or fragment thereof) from incompatibility with container materials and closure systems. In some embodiments, the suspended ALD-coated microparticles have reduced incompatibility with syringe lubricants (e.g., silicone oil), interfaces presented by air bubbles, and surfaces present in glass or polymer containers, as well as polymer surfaces such as vial stoppers and syringe plungers. In other embodiments, the suspended ALD-coated microparticles disclosed herein can be suspended in an isotonic buffer to make them suitable for immediate parenteral administration to a subject at the point of care. In other embodiments, the suspended ALD-coated microparticles of the suspension disclosed herein can be a stable liquid formulation that reduces or eliminates cold chain requirements for storage and transportation of the target antigen or vaccine.According to these embodiments, the formulation for suspending the metal oxide coated antigen particles may include a histidine buffer, a histidine-like buffer, or other non-chelating buffer. In certain embodiments, the non-chelating buffer (e.g., histidine) may have a pH of about 4.0 to about 8.0, or about 5.0 to about 7.5, or other suitable pH.

[0031] In certain embodiments, the suspension formulation for the coated microparticles disclosed herein may contain histidine, imidazole, glycine, bistrismethane, tris, bicine, glycylglycine, or similar non-chelating organic buffers, or other agents or buffers known to those skilled in the art as Good's or amine buffers. In certain embodiments, the concentration of the buffer or agent used as the suspension formulation disclosed herein may be reduced (e.g., 20 mM or less) and may further contain phosphate (e.g., sodium phosphate or potassium phosphate). According to these embodiments, the concentration of histidine and / or phosphate may be about 40 mM or less; or about 30 mM or less; about 20 mM or less; about 10 mM or less; or about 5 mM or less. In some embodiments, the suspension formulation for the coated microparticles disclosed herein may contain sodium phosphate at a concentration of about 5 mM or less. In other embodiments, the suspension formulation for the coated microparticles disclosed herein may contain histidine at a concentration of about 5 mM or less. According to these embodiments, the pH of the suspension formulation comprising histidine and / or phosphate is less than about pH 8.0 when the concentration of histidine and / or phosphate is about 20.0 mM or less. In some embodiments, the pH of the suspension formulation comprising histidine and / or phosphate is less than about pH 8.0 when the concentration of histidine and / or phosphate is about 15.0 mM or less. In some embodiments, the pH of the suspension formulation comprising histidine and / or phosphate is less than about pH 8.0 when the concentration of histidine and / or phosphate is about 10.0 mM or less. In some embodiments, the pH of the suspension formulation comprising histidine and / or phosphate is less than about pH 8.0 when the concentration of histidine and / or phosphate is about 5.0 mM or less. In some embodiments, the pH of the suspension formulation comprising histidine and / or phosphate is greater than about pH 7.0 when the concentration of histidine and / or phosphate is about 20.0 mM or less.In some embodiments, the pH of the suspension formulation containing histidine and / or phosphate is about pH 7.0 or more when the concentration of histidine and / or phosphate is about 15.0 mM or less. In other embodiments, the pH of the suspension formulation containing histidine and / or phosphate is about pH 7.0 or more when the concentration of histidine and / or phosphate is about 10.0 mM or less. In some embodiments, the pH of the suspension formulation containing histidine and / or phosphate is about pH 7.0 or more when the concentration of histidine and / or phosphate is about 5.0 mM or less. In certain embodiments, the pH of the suspension formulation containing phosphate is about 6.0 to about 8.0; or about 6.0 to about 7.5 when the concentration of phosphate is about 20.0 mM or less; or 15 mM or less; or 10 mM or less; or 5 mM or less. In certain embodiments, the pH of the histidine-containing suspension formulation is about 7.0 or greater, or about 7.0 to about 8.5, or about 7.5 to about 8.5 when the concentration of histidine is less than 20 mM; or 15 mM or less; or 10 mM or less; or 5 mM or less. In certain embodiments, these suspension formulations can further comprise one or more additional agents to stabilize and / or facilitate delivery of the coated microparticles containing one or more antigens.

[0032] In some embodiments, the suspension formulation for the coated antigen-containing microparticles may contain histidine and may be stored at any temperature for storage, transportation, and delivery to a subject. According to these embodiments, the suspension formulation containing histidine may be stored at temperatures below about 4°C to above about 60°C, or above about 50°C, for a period of several hours to about one week, one month, two months, or more, without premature or unplanned release of one or more coated antigens. In some embodiments, these suspension formulations do not contain phosphate. In certain embodiments, these suspension formulations may further include one or more additional agents (e.g., trehalose) for stabilizing and / or facilitating delivery of the coated microparticles containing one or more antigens. In some embodiments, the suspension formulation for the coated antigen-containing microparticles can include histidine and trehalose (or sucrose or similar agent) and can be stored at temperatures below about 4° C. to above about 60° C., or above about 50° C., for a period of several hours to about a week, a month, two months or more without premature or unscheduled release of one or more coated antigens, enhancing storage, transport and delivery of the coated antigen-containing microparticles. In certain embodiments, the formulations containing histidine do not contain phosphate. In other embodiments, the suspension formulations disclosed herein do not contain aqueous phosphate, citrate, or sulfate. In other embodiments, the suspension formulations disclosed herein can further include a salt at a concentration of less than about 0.200 M, or less than or equal to about 0.150 M or about 0.100 M.

[0033] In certain embodiments, the suspension formulations disclosed herein may further include trehalose or sucrose or other agents. According to these embodiments, disaccharide agents such as trehalose or sucrose, or other equivalent disaccharides or sugars, may be used to achieve isotonicity of the composition. In certain embodiments, the concentration of trehalose and / or sucrose may be about 20% w / v or less. In certain embodiments, the concentration of trehalose and / or sucrose may be about 15% w / v or less. In other embodiments, the suspension formulations may further include one or more salts (e.g., sodium salt, potassium salt). In certain embodiments, the polyvalent anionic agent may be present at a concentration of about 50 mM or less, while the amine-based agent (e.g., histamine) or buffering agent may be present at a concentration of about 100 mM or less in the suspension formulations provided herein. In still other embodiments, the suspension formulations disclosed herein may include a surfactant, such as a polysorbate.

[0034] In some embodiments, the suspension formulations disclosed herein do not include a buffer containing citrate, phosphate, or a salt thereof such as sodium. In certain embodiments, the suspension formulations disclosed herein do not include a buffer containing citrate. In certain embodiments, the suspension formulations disclosed herein do not include a buffer containing citrate-phosphate formulations. According to these embodiments, citrate alone or in combination with phosphate induces instability of the coated antigen microparticles disclosed herein. In other embodiments, the suspension formulations disclosed herein do not include a buffer containing a phosphate buffer. In certain embodiments, the suspension formulations disclosed herein do not include a buffer containing a sodium phosphate formulation. According to these embodiments, buffers containing citrate, phosphate, or chloride were particularly influential in imparting deleterious changes to the particles. The suspension and administration of the coated antigen microparticles disclosed herein requires the selection of a diluent for injection to avoid stability problems in the final product. In certain embodiments, the suspension formulations disclosed herein should avoid multivalent anions (e.g., citrate, phosphate, sulfate) to maintain the integrity of the microparticles.

[0035] In other embodiments, the compositions may include one or more surfactants, such as non-ionic surfactants (e.g., polysorbate 20, polysorbate 80, poloxamer 188), or another similar agent. In other embodiments, the compositions disclosed herein may include antimicrobial agents or preservatives (e.g., benzyl alcohol, methylparaben, creosol, phenol, etc.). In other embodiments, when preservatives and antimicrobial agents are added to the formulations used to suspend the metal oxide-coated antigen microparticles, the target antigens protected by the metal oxide coating may be further protected from adverse reactions to the additives by suspending the metal oxide-coated antigens in low ionic strength non-chelating buffers disclosed herein. In accordance with these embodiments, surfactants, antimicrobial agents, and preservatives may be added to these formulations without damaging the antigens embedded within the metal oxide-coated microparticles. In certain embodiments, the suspension formulations or compositions disclosed herein include low ionic strength non-chelating buffers and further include one or more of a preservative or surfactant or other suitable storage agent. According to these embodiments, the resulting suspension is stable while reducing or preventing release of antigen from the coated microparticles into the suspension formulation prior to delivery to a subject.

[0036] Formulation of vaccines and therapeutic proteins in dry form (e.g., made by lyophilization or spray drying) can mitigate some of the potential damage to vaccines and protein therapeutics caused by exposure to various interfaces (e.g., silicone oil-water interface, air-water interface, container-water interface), preservatives and antimicrobial agents. In some embodiments, the compositions and methods disclosed herein relate to embedding or encapsulating vaccines or therapeutic proteins within a dry powder matrix or within a lyophilized formulation cake to reduce or prevent direct contact with container stoppers, seals, plungers, and lubricants. According to these embodiments, some interferences are mitigated, but formulations in dry form may pose additional challenges. In other embodiments, filling vials with dry powders is more difficult than filling with liquid formulations, and dry powders and lyophilized formulations must still be suspended in a vehicle or formulation prior to injection. The embodiments disclosed herein take advantage of the stable nature of metal oxide coated antigen microparticles while formulating these microparticles into liquid or aqueous suspensions for easy distribution, storage, transportation, and use.

[0037] In some embodiments, the antigens disclosed herein with metal oxide coating can be produced by a heat stabilization process that produces microparticles of antigen and at least one adjuvant embedded in a glassy sugar matrix. According to these embodiments, atomic layer deposition can be used to coat glassy particles and create stable microparticles with a defined atomic layer of metal oxide that dissolves over a specific period of time, on which a priming layer of antigen and potentially one or more boost layers are included. In certain embodiments disclosed herein, non-chelating buffers can be used to suspend the microparticles for later use without compromising the integrity of the microparticles and to reduce premature release of antigen.

[0038] Certain embodiments of the present disclosure provide methods for making immunogenic agent-containing particles for use in the suspension formulations disclosed herein, which may include combining at least one immunogenic agent with at least one glass forming agent to form a first liquid immunogenic composition, dehydrating the first liquid immunogenic composition to form immunogenic agent-containing glass microparticles, and coating the immunogenic agent-containing glass microparticles with one or more outer coating layers. In some embodiments, the first liquid immunogenic composition may be dehydrated by lyophilization, vacuum drying, spray drying, or spray freeze drying.

[0039] According to these embodiments, the at least one immunogenic agent can include one or more antigens, such as viral antigens, bacterial antigens, toxins, or combinations thereof. In some embodiments, the at least one immunogenic agent can also include, but is not limited to, recombinant peptides, recombinant proteins, peptides derived from target proteins or pathogens, synthetic peptides or proteins, virus-like particles, live viruses, live, attenuated live viruses, inactivated viruses, or combinations thereof.

[0040] In certain embodiments, the at least one immunogenic agent that may be coated with a metal oxide in the formulation may include one or more antigens, such as antigens from human papillomavirus, ricin toxin, anthrax, botulinum, Ebola virus, poliovirus, norovirus, rotavirus, hepatitis C, chickenpox, herpes simplex, cytomegalovirus, Japanese encephalitis, dengue virus, West Nile virus, Zika virus, Yersinia, Streptococcus pneumoniae, Salmonella, Clostridium difficile, or combinations thereof. In certain embodiments, the at least one immunogenic agent may be a multimeric complex.

[0041] In certain embodiments, the pathogenic virus is, for example, a papovavirus (e.g., a papillomavirus, including human papillomavirus (HPV)), a herpesvirus (e.g., herpes simplex virus, varicella zoster virus, bovine herpesvirus-1, cytomegalovirus), a poxvirus (e.g., smallpox virus), a reovirus (e.g., rotavirus), a parvovirus (e.g., parvovirus B19, canine parvovirus), a picornavirus (e.g., poliovirus, hepatitis A), a togavirus (e.g., an alphavirus, such as rubella virus, chikungunya virus), a hepadnavirus (e.g., hepatitis B virus), a flavivirus (e.g., dengue virus, The virus may be an influenza virus, hepatitis C virus, West Nile virus, yellow fever virus, Zika virus, Japanese encephalitis virus), orthomyxovirus (e.g., influenza A virus, hepatitis B virus, influenza C virus), paramyxovirus (e.g., measles virus, mumps virus, respiratory syncytial virus, canine distemper virus, parainfluenza virus), rhabdovirus (e.g., rabies virus), filovirus (e.g., Ebola virus), SARS, or coronavirus (e.g., COVID19, delta, mumps, or other strains), or a combination thereof, or a polypeptide or polynucleotide thereof provided herein.

[0042] In other embodiments, the pathogen is selected from the group consisting of Pasteurella haemolytica, Clostridium difficile, Clostridium haemolyticum, Clostridium tetani, Corynebacterium diphtheria, Neorickettsia resticii, Streptococcus equi, Streptococcus pneumoniae, Salmonella spp., Chlamydia trachomatis, Bacillus anthracis, Yersinia spp. The toxin may be a bacterium or a bacterial toxin, including, but not limited to, Bacillus subtilis spp., and Clostridium botulinum, or a combination thereof.

[0043] In some embodiments, the pathogen is selected from the group consisting of Cryptococcus spp. (e.g., neoformans and gattii), Aspergillus spp. (e.g., fumigatus), Blastomyces spp. (e.g., dermatitidis), Candida albicans, Paracoccidioides spp. (e.g., brasiliensis), Sporothrix spp. (e.g., schenkii and brasiliensis), Histoplasma capsulatum, and the like. The fungus may be, but is not limited to, Pneumocystis capsulatum, Pneumocystis jirovecii, and Coccidioides immitis, or a combination thereof.

[0044] In yet other embodiments, the antigen can include a pathogen, such as a toxin, such as ricin toxin or botulinum toxin or anthrax toxin or another toxin. In some embodiments, the immunogenic agent-containing particles described herein can be used to manufacture one or more immunogenic compositions for use as vaccines for animals, such as household pets. According to these embodiments, the immunogenic compositions can be administered to, for example, canines, felines, equines, bovines, hircines, caprines, or poultry (e.g., chickens, turkeys, ducks, geese).

[0045] In certain embodiments, the immunogenic agent-containing particles described herein can be used to generate one or more immunogenic compositions for administration to a dog to reduce or prevent an infection or to treat an infection, including, but not limited to, immunogenic compositions related to canine parvovirus (CPV), canine distemper virus (CDV), canine adenovirus (CAV), rabies, canine parainfluenza virus (CPiV), canine influenza virus, canine coronavirus, measles virus, Bordetella bronchiseptica, Leptospira spp., and Borrelia burgdorferi, or combinations thereof.

[0046] In some embodiments, the immunogenic agent-containing particles described herein can be used to produce one or more immunogenic compositions for administration to a cat to reduce or prevent an infection or to treat an infection, including, but not limited to, immunogenic compositions related to feline herpesvirus type 1 (FHV1), feline calicivirus (FCV), feline panleukopenia virus (FPV), rabies virus, feline leukemia virus (FeLV), feline immunodeficiency virus, pathogenic systemic feline calicivirus, feline chlamydia, Pasteurella haemolytica, and Bordetella bronchiseptica, or combinations thereof.

[0047] In other embodiments, the immunogenic agent-containing particles described herein can be used to generate one or more immunogenic compositions for administration to horses to reduce or prevent infection or to treat an infection, including, but not limited to, immunogenic compositions associated with Eastern Equine Encephalomyelitis Virus, Western Equine Encephalomyelitis Virus, Venezuelan Equine Encephalomyelitis Virus, Bovine Papilloma Virus, Rabies Virus, Clostridium tetani, West Nile Virus, Equine Influenza Virus, Potomac Fever (Neorickettsia risticii), Streptococcus equi, and Rhinopneumonitis (Equine Herpesvirus Type 1), or combinations thereof.

[0048] In certain embodiments, the immunogenic agent-containing particles described herein can be used to generate one or more immunogenic compositions for administration to cattle to reduce or prevent infection or to treat an infection, including, but not limited to, immunogenic compositions associated with bovine rhinotracheitis (IBR), parainfluenza type 3 (PI3), bovine viral diarrhea (BVD), bovine respiratory syncytial virus (BRSV), blackleg disease (Clostridium chauvoei), malignant edema (Clostridium septicum), infectious necrotic hepatitis (Clostridium novyi), enterotoxemia (Clostridium perfringens types C and D), Pasteurella haemolytica, and flushing (Clostridium haemolyticum), or combinations thereof.

[0049] In some embodiments, the immunogenic agent-containing particles described herein can be used to generate one or more immunogenic compositions for administration to poultry to reduce or prevent infection or to treat an infection, including, but not limited to, immunogenic compositions related to Marek's disease (Marek's disease virus), tenosynovitis (reovirus), encephalomyelitis (avian encephalomyelitis virus), fowl pox (avipox virus), chicken infectious anemia (chicken anemia virus), fowl cholera (Pasteurella multocida), Newcastle disease / infectious bronchitis (Newcastle disease virus), Riemerella anatipestifer, duck viral hepatitis (duck hepatitis virus), and duck viral enteritis (duck herpesvirus type 1), or combinations thereof.

[0050] In some embodiments, the immunogenic agent-containing particles described herein can be used to generate immunogenic compositions used for administration to humans. In certain embodiments, the immunogenic agent-containing particles described herein can be used to deliver one or more immunogenic compositions to a human infant or child or adolescent, including, but not limited to, vaccines for varicella zoster (chickenpox), diphtheria, Haemophilus influenzae type b (Hib), hepatitis A, hepatitis B, influenza, measles, mumps, whooping cough, polio, pneumococcal disease, rotavirus, rubella, and tetanus. In other embodiments, the immunogenic agent-containing particles described herein may be used to deliver one or more immunogenic compositions to a pre-teen or teenage human, including, but not limited to, vaccines for influenza, tetanus, diphtheria, whooping cough, human papilloma virus, meningococcal disease, hepatitis B, hepatitis A, polio, measles, mumps, rubella, and varicella zoster. In yet other embodiments, the immunogenic agent-containing particles described herein may be used to deliver one or more immunogenic compositions to a human adult, including, but not limited to, immunogenic compositions for influenza (e.g., types A, B, or C), tetanus, diphtheria, whooping cough, shingles, pneumococcal disease, meningococcal disease, measles, mumps, rubella, chickenpox, hepatitis A, hepatitis B, and Haemophilus influenzae type b.

[0051] In other embodiments, the immunogenic agent-containing particles described herein may be used to generate immunogenic compositions useful for administration to humans, including, but not limited to, immunogenic compositions against travel-related diseases, including, but not limited to, hepatitis A, hepatitis B, typhoid, paratyphoid, meningococcal disease, yellow fever, dengue fever, rabies virus, chikungunya, and Japanese encephalitis.

[0052] In still other embodiments, the immunogenic agent-containing particles described herein may be used to generate immunogenic compositions used to administer to humans, including, but not limited to, immunogenic compositions against human papillomavirus (e.g., HPV16, HPV18, HPV31, HPV45, or HPV6 or HPV11, or any other HPV), herpes simplex virus, smallpox virus, rotavirus, parvovirus B19 vaccine, chikungunya virus, dengue virus (e.g., dengue-1, dengue-2, dengue-3, or dengue-4), hepatitis C virus, West Nile virus, Zika virus, respiratory syncytial virus, rabies virus, and Ebola virus.

[0053] In some embodiments, the at least one glass forming agent can include at least one of trehalose, sucrose, ficoll, dextran, sucrose, maltotriose, lactose, mannitol, hydroxyethyl starch, glycine, cyclodextrin, povidone, and the like. In certain embodiments, the at least one glass forming agent can include trehalose, sucrose, or hydroxyethyl starch. In certain embodiments, the at least one glass forming agent can include trehalose. According to this embodiment, trehalose can be included as a glass forming agent and may be present in the primary liquid immunogenic composition at a weight to volume (w / v) concentration of about 0.1% to about 40%, about 1% to about 30%, about 5% to about 20%, or about 8% to about 15%.

[0054] In other embodiments, the glass forming agent also includes at least one smoothing excipient. According to these embodiments, the smoothing excipient can be hydroxyethyl starch or other pharmacologically acceptable plasma expanders, such as human serum albumin (HSA), other serum albumins, dextran, hetastarch, plasma protein factors, etc., or combinations thereof. In certain embodiments, the smoothing excipient can also be a primary glass forming agent. In some embodiments, the smoothing excipient is hydroxyethyl starch. In some embodiments, the smoothing agent disclosed herein can be a primary glass forming agent. According to these embodiments, the smoothing excipient can be present in the primary immunogenic composition at a weight to volume (w / v) concentration of about 0.1% to about 40%, about 1% to about 30%, about 5% to about 20%, or about 8% to about 15%. In certain embodiments, the smoothing excipient may be different from the first glass forming agent, and the smoothing excipient may be present in the first immunogenic composition at a weight to volume (w / v) concentration of about 0.1% to about 10%, about 0.1% to about 5%, about 0.1% to about 2.5%, about 0.1% to about 0.5%. In certain embodiments, the glass forming agent present in the immunogenic composition is trehalose and the smoothing excipient for the particles is hydroxyethyl starch.

[0055] In some embodiments, each layer of the one or more outer metal oxide coating layers can comprise aluminum oxide, aluminum alkoxide (e.g., alkone), silicon dioxide (SiO2), titanium dioxide (TiO2), silicon nitride (Si3N4), zinc oxide (ZnO), zircon (MLD), zirconia, hafnium oxide, alone or in suitable combination compositions, or other suitable (e.g., biocompatible) metal oxide coatings. According to these embodiments, the outer coating layer can be about 0.1 nm to about 20 nm thick. In certain embodiments, the immunogenic agent-containing particles can comprise a sufficient number of outer coating layers to delay or provide a timed release of at least one immunogenic agent from the central or innermost immunogenic agent-containing glassy microparticle.

[0056] In certain embodiments, one or more coating layers disclosed herein can function as an adjuvant to enhance the immune response in a subject to one or more immunogenic agents of the immunogenic agent-containing particle.In certain embodiments, one or more coating layers can contain a concentration that can induce a rapid immune response to one or more immunogenic agents of the immunogenic agent-containing particle.

[0057] In other embodiments, the immunogenic agent-containing particle may further comprise at least a second immunogenic agent deposited as a layer on the outermost coating layer of the immunogenic agent-containing particle. According to these embodiments, the layer of at least a second immunogenic agent may be embedded in a glassy matrix of at least a second glass-forming agent to stabilize the second immunogenic agent.

[0058] In other embodiments, methods are also provided for producing at least one layer of at least a second immunogenic agent, and embedding the second immunogenic agent in the glassy matrix of at least a second glass forming agent.In some embodiments, at least one additional outer coating layer covers or encases the layer of at least a second immunogenic agent.In certain embodiments, the second immunogenic agent is the same as the at least one immunogenic agent of the central immunogenic agent-containing glassy microparticle.In other embodiments, the second immunogenic agent is different from the at least one immunogenic agent of the central immunogenic agent-containing glassy microparticle.

[0059] In some embodiments, suspensions containing the immunogenic agent-containing particles described herein can be stored for extended periods of time without refrigeration at temperatures up to about 50° C. to about 60° C. In certain embodiments, the immunogenic agent-containing particles described herein can be stored at about 50° C. to about 60° C. without refrigeration for up to about 3 months, or up to about 4 months, or up to about 6 months, or up to about 9 months, or up to about 12 months, or up to about 15 months, or up to about 18 months, or up to about 24 months, or longer, without negative effects on the immunogenic agent-containing particles (e.g., degradation).

[0060] Other embodiments of the present disclosure provide immunogenic compositions comprising a plurality of immunogenic agent-containing particles as described herein.In certain embodiments, the immunogenic composition can comprise immunogenic agent-containing particles in a pharma-ceutically acceptable excipient to produce a pharma-ceutically acceptable immunogenic composition.According to these embodiments, the described immunogenic compositions can induce an immune response against the immunogenic agent when administered to a subject.

[0061] In some embodiments, the immunogenic composition is a single-dose immunogenic composition, comprising a prime dose and a boost dose of at least one immunogenic agent. According to these embodiments, the prime dose and the boost dose of at least one immunogenic agent can be in the same immunogenic agent-containing particle, or in separate immunogenic agent-containing particles. When in separate particles, the at least one immunogenic agent of the prime dose can be sequestered in the immunogenic agent-containing glass microparticle that does not have any outer coating layer, or in the immunogenic agent-containing particle, while the at least one immunogenic agent of the boost dose is in another immunogenic agent-containing particle. Whether the priming dose is in an immunogenic agent-containing glass microparticle or in an immunogenic agent-containing particle is determined by whether an immediate or delayed response is desired, where the outer coating layer of the immunogenic agent-containing particle that sequesters the priming dose temporarily delays the release of the priming dose, thus delaying exposure of the priming dose to the subject.

[0062] In some embodiments, the immunogenic composition can be a single-dose immunogenic composition that can induce immune response against two or more different immunogenic agents.According to these embodiments, two or more different immunogenic agents can be contained in the same immunogenic agent-containing particle or in separate immunogenic agent-containing particles.In other embodiments, when two or more different immunogenic agents are contained in separate immunogenic agent-containing particles, each different immunogenic agent-containing particle contains a different immunogenic agent or combination of immunogenic agents.

[0063] In some embodiments, the immunogenic composition may include a priming dose and a boosting dose of a first immunogenic agent and a priming dose and a boosting dose of a second immunogenic agent. According to these embodiments, the priming dose and the boosting dose of the first immunogenic agent are in the first immunogenic agent-containing particle, while the priming dose and the boosting dose of the second immunogenic agent are sequestered in the second immunogenic agent-containing particle. In other embodiments, the priming dose and boost dose of a first immunogenic agent are located in a first pair of particles, with the priming dose being in a separate particle from the boosting dose, the priming dose and boost dose of a second immunogenic agent are sequestered in a second pair of particles, with the priming dose being in a separate particle from the boosting dose, the priming dose for each of the first and second immunogenic agents being in separate immunogenic agent-containing glass microparticles, and the boosting dose for each of the first and second immunogenic agents being in separate immunogenic agent-containing particles. In yet other embodiments, the priming dose and boost dose of a first immunogenic agent are sequestered in a first pair of immunogenic agent-containing particles, the priming dose being in a separate particle from the boosting dose, the priming dose and boosting dose of a second immunogenic agent are located in a second pair of immunogenic agent-containing particles, the priming dose being in a separate particle from the boosting dose, the priming dose for the first immunogenic agent is in an immunogenic agent-containing glass microparticle, the priming dose for the second antigen is in a separate immunogenic agent-containing particle, and the boost dose for each of the first antigen and second antigen is in a separate immunogenic agent-containing particle.

[0064] In other embodiments, the immunogenic composition can include a standard vaccine composition and a plurality of immunogenic agent-containing particles as described, where at least one immunogenic agent elicits a boost immune response to the standard vaccine composition.

[0065] In yet other embodiments, the immunogenic composition may comprise a plurality of first immunogenic agent-containing particles described herein, where the first immunogenic agent-containing particles may comprise at least a first immunogenic agent, a plurality of second immunogenic agent-containing particles described herein, where the second immunogenic agent-containing particles comprise at least a second immunogenic agent different from the first immunogenic agent, and a pharma- ceutically acceptable excipient. According to an embodiment, the immunogenic composition may further comprise a plurality of at least one additional immunogenic agent-containing particles described herein, where the at least one additional immunogenic agent-containing particle comprises at least one additional immunogenic agent that is not the first immunogenic agent or the second immunogenic agent.

[0066] Other embodiments provide methods for eliciting an immune response in a subject, which may include administering to the subject an immunogenic composition described herein. According to these embodiments, the immunogenic composition is capable of inducing an immune response in the subject. The immune response induced by the immunogenic composition may be prophylactic or therapeutic, depending on the immunogenic agent.

[0067] Other embodiments disclosed herein relate to kits for storage, transport and use of the suspended metal oxide coated antigens disclosed herein. In certain embodiments, the kits can include ready-to-use syringes containing the suspension formulations disclosed herein for delivering doses of target antigens to subjects. In other embodiments, the kits can include multi-dose containers containing the suspension formulations disclosed herein for long-term storage, reducing cryopreservation requirements, reliable dosing, and reducing interference with storage components such as stoppers, oils, and preservatives. In accordance with these embodiments, a single vial of multiple doses containing a desired antigen useful for treating, reducing the onset of, or preventing a health condition can be used to treat multiple subjects, including human subjects and animals (e.g., livestock).

[0068] In some embodiments, the formulations disclosed herein can include metal oxide coated antigen microparticles, a histidine buffer at a pH of about 6.0 to about 8.0, and, optionally, one or more preservatives and one or more surfactants. According to these embodiments, the histidine buffer can be about 1 mM to about 50 mM.

[0069] Pharmaceutical preparations suitable for injection include sterile aqueous solutions and dispersions. The carrier can be a solvent or dispersion medium, for example, containing water, saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof.

[0070] In some embodiments, certain additives that enhance the stability, sterility, and isotonicity of the compositions, including antimicrobial preservatives, antioxidants, and buffers, may be added to the formulations disclosed herein to improve stability and reduce contamination. In some embodiments, antibacterial and antifungal agents (e.g., benzyl alcohol, methylparaben, paraben, chlorobutanol, phenol, sorbic acid, cresol, metacresol, etc.) may be added to reduce bacterial contamination in suspensions for parenteral or other modes of administration.

[0071] Sterile injectable solutions can be prepared by incorporating the suspensions utilized in carrying out the present disclosure in the required amount of the appropriate solvent with a certain amount of other ingredients, as required. Such compositions can be mixed with a suitable carrier, diluent, or excipient (e.g., sterile water, saline, glucose, dextrose, etc.).

[0072] The compositions of the present invention can be provided as liquid preparations (e.g., isotonic aqueous solutions, suspensions, emulsions, or viscous compositions) that can be buffered to a selected pH. The selection of suitable carriers and other additives can depend on the route of administration and the nature of the particular dosage form, for example, the form of the liquid dosage form (e.g., whether the composition is formulated into a solution, suspension, gel, or another liquid form, such as a time-release form or a liquid-filled form). Solutions, suspensions, and gels usually contain a major amount of water (e.g., purified sterile water) in addition to suspended particles or suspended particulates. Small amounts of other components, such as pH adjusters (e.g., bases such as NaOH), emulsifiers or dispersants, buffers, preservatives, wetting agents, and gelling agents (e.g., methylcellulose), can also be present. The compositions can be isotonic, i.e., they can have the same osmotic pressure as blood and tears.

[0073] In some embodiments, the desired isotonicity of the compositions of the present disclosure can be achieved using sodium chloride, or other pharma- ceutically acceptable agents (e.g., dextrose, boric acid, sodium tartrate, propylene glycol, or organic solutes). The viscosity of the composition can be maintained at a selected level, if desired, using a pharma- ceutically acceptable thickening agent. Methylcellulose is readily and economically available and easy to handle. Other suitable thickening agents include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, and the like. The concentration of the thickening agent depends on the agent selected. The key is to use an amount that can obtain the selected viscosity. Viscous compositions are usually prepared from solutions by the addition of such thickening agents.

[0074] The compositions can be administered in dosages and by techniques well known to those of ordinary skill in the medical and veterinary arts, taking into account factors such as the age, sex, weight, and condition of the particular patient, as well as the composition (e.g., liquid) used for administration. Dosages for humans or other mammals can be determined without undue experimentation by those of ordinary skill in the art from this disclosure and knowledge in the art.

[0075] In some embodiments, the kit is intended for use in storing and transporting the compositions disclosed herein in either a dry or suspended state and includes at least one container. In certain embodiments, the kit may include at least one composition of metal oxide-coated microparticles of a target antigen in a dry form, and a buffer solution including a non-chelating buffer solution for suspension of the microparticles. In other embodiments, the kit may include a ready-to-use suspension of metal oxide-coated microparticles of at least one target antigen (or combination of target antigens) in a single or multi-dose container such as a vial or syringe. In certain embodiments, the non-chelating buffer solution includes histidine.

[0076] In some embodiments, the kit is a vaccine kit of ready-to-use suspended metal oxide coated microparticles of target antigens for use in reducing or preventing the onset of infection with a microbial agent such as a virus. In certain embodiments, the antigens of any of the formulations disclosed herein may include, but are not limited to, polypeptides (e.g., recombinant, chimeric, or naturally occurring), polynucleotides (RNA, DNA, or hybrid molecules), small molecules, microbial agents such as viruses or bacteria, fungi, prions, toxins, or other antigens.

[0077] In some embodiments, the kit can include instructions for use according to any of the methods described herein. The instructions found in the kit can include instructions for administering the formulation and optionally a second therapeutic agent to treat, delay the onset, or alleviate the target condition as described herein. The kit can further include instructions for selecting an individual suitable for treatment based on identifying whether the individual has or is suspected of developing the target condition. In yet other embodiments, the instructions can include instructions for administering a pre-filled, ready-to-administer syringe of the vaccine formulation disclosed herein to a subject at risk of developing a disease or condition disclosed herein.

[0078] In some embodiments, the instructions for use of the ready-to-use antigen-containing formulation generally include information including, but not limited to, dosage and treatment schedule. The containers of the kit can include unit dosages or bulk packages (e.g., multi-dose packages) or sub-unit doses. The kit can further include a delivery device, such as a syringe, an implant device or other timed delivery device. The instructions provided in the kit of the present invention are typically written instructions on a label or insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions carried on a magnetic or optical memory disk) are also acceptable.

[0079] In some embodiments, the kit may be in a suitable package. Suitable packages include, but are not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar® bags or plastic bags), and the like. Packages for use in combination with specific devices, such as inhalers, nasal administration devices (e.g., atomizers), or injection devices, such as mini-pumps, are also contemplated. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle). The kits contemplated herein may contain at least one active agent in a composition, such as one or more antigens coated with metal oxide or ALD coated as described herein.

[0080] The kit may optionally provide additional components, such as buffers and interpretive information. Typically, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiments, the invention provides an article of manufacture comprising the contents of the above-described kit. EXAMPLES

[0081] The following examples are included to illustrate certain embodiments and are not to be considered as limiting the present disclosure. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques that have been discovered to function in the implementation of the claimed methods, compositions and devices. However, those skilled in the art should understand in light of this disclosure that modifications can be made in some of the disclosed embodiments and examples without departing from the spirit and scope of the present invention and still obtain the same or similar results.

[0082] Example 1 In one exemplary method, the colloidal stability of atomic layer deposition coated particles in inorganic (e.g., phosphate) and organic (e.g., histidine) buffers was investigated for the stability and integrity of suspended microparticles. Spray-dried particles in a formulation of trehalose, histidine, ammonium acetate, and polysorbate 20 were coated with (e.g., 250) molecular layers of alumina in an atomic layer deposition reactor. The resulting particles were suspended in buffers of various pHs containing approximately 9.5% trehalose and either 5 mM histidine, 20 mM histidine, or 5 mM sodium phosphate. The particles were suspended to a concentration of approximately 1.0 mg / ml. Zeta potential was assessed. Zeta potential is a measure of colloidal stability that is often used to predict interactions within therapeutic formulations. A formulation is considered unstable if the zeta potential is between 20 and -30 mV. Intermediate stability is indicated by a value of -30 to -40 mV. Good stability is indicated by stability between -40 and -60 mV. Excellent stability is indicated by values ​​below -60 mV. Particles in 20 mM histidine were colloidally unstable at all pH values ​​tested, while particles suspended in histidine buffer at the lowest concentrations, such as 5 mM histidine, were unstable only at pH 7 and below. In contrast, 5 mM sodium phosphate showed good stability at pH 6 to 7.5. (See, for example, Figure 1).

[0083] Example 2 In another exemplary method, the addition of polysorbate 80 to the suspension medium does not significantly change the release profile and maintains the integrity of the antigen-containing particles. In this method, spray-dried particles containing an exemplary antigen (e.g., ovalbumin) labeled with a dye that fluoresces in the infrared (IR-OVA), trehalose, histidine, ammonium acetate, and polysorbate 20 were coated with a molecular layer of alumina in an atomic layer deposition reactor. The coated particles were suspended in approximately 9.5% trehalose, 5 mM histidine, 150 mM sodium chloride buffer with or without 0.04% polysorbate 80, and incubated at either room temperature or 50°C for one week. After incubation, the samples were centrifuged at 16,000 x g for 5 minutes and the supernatant was decanted. The remaining pellet was dissolved in citrate-phosphate buffer to remove the alumina coating and to assess the remaining protein. The dissolved pellet was analyzed for IR-ovalbumin by measuring absorbance at 672 nm. Additionally, the decanted suspension buffer was analyzed on an SDS-PAGE gel and any bands were quantified. Minimal release of IR-OVA from the particles into the suspension medium was observed via SDS-PAGE gel. Addition of polysorbate 80 did not significantly alter protein release (see, e.g., Figures 2A and 2B).

[0084] FIG. 2A represents an analysis of IR dye-labeled ovalbumin released from ALD-coated powder. Particles were initially suspended in 9.5% trehalose, 5 mM histidine, 150 mM sodium chloride for 1 week. Light gray bars represent the amount of protein released into the suspension medium after 1 week of incubation. Dark bars represent the protein released from the particles after the particles were collected by centrifugation and the ALD coating was removed by dissolving in citrate-phosphate buffer. Light gray bars represent the amount of protein released into the suspension medium after 1 week of incubation.

[0085] FIG. 2B depicts an exemplary plot of the release of IR-labeled ovalbumin from ALD-coated microparticles after incubation for 1 week at room temperature or 50° C. in 9.5% trehalose, 5 mM histidine, 150 mM sodium chloride in the presence and absence of 0.05% polysorbate 80.

[0086] Example 3 In another example, spray-dried particles containing an exemplary antigen (e.g., ovalbumin) labeled with an infrared-fluorescing dye (IR-OVA), trehalose, histidine, ammonium acetate, and polysorbate 20 were coated with a molecular layer of metal ions (e.g., alumina) in an atomic layer deposition reactor. The coated particles were suspended in about 9.5% trehalose with 5 mM sodium phosphate, 5 mM histidine, or no buffering agent, and incubated at room temperature or 50° C. for one week. After incubation, the samples were centrifuged at 16,000×g for 5 minutes and the suspension buffer was removed. The suspension buffer and dissolved pellet were analyzed for IR-ovalbumin release by measuring absorbance at 672 nm. Additionally, the suspension buffer was analyzed on an SDS-PAGE gel and any bands were quantified. Inclusion of phosphate as a resuspension buffer resulted in the release of protein into the suspension buffer during the incubation period. In samples lacking buffers (no histidine or phosphate), the level of released protein was minimal. In contrast, samples containing histidine at lower concentrations had no detectable protein release in suspension buffer, regardless of incubation temperature. These experiments support the use of non-chelating organic buffers for long-term maintenance of antigen integrity, including metal oxide antigens, in ready-to-use or easily hydratable formulations. In addition, these non-chelating organic buffer formulations are designed for use in therapeutic delivery devices to reduce interference caused by preservatives, such as antimicrobial agents, oils, silicones, waxes, or other ingredients used to ensure closure of containers used to store therapeutic agents, and to provide ready-to-use single or multiple dose formulations for use over a day or weeks and even months (e.g., in remote areas for use in humans to treat two or more subjects using the same vial or in pets or livestock). (See, e.g., Figure 3).

[0087] FIG. 3 presents a plot of the release of IR-labeled ovalbumin after suspension in 9.5% trehalose solution containing phosphate, histidine, or no buffer, followed by incubation at room temperature or at 50° C. for 1 week.

[0088] Example 4 In another exemplary experiment, coated particles containing IR-labeled ovalbumin (IR-OVA, an exemplary antigen) were suspended in a buffer containing either 9.5% trehalose, 5 mM histidine or 9.5% trehalose, 5 mM phosphate and incubated at either room temperature or 50° C. for up to 8 weeks. After incubation, samples were centrifuged at 16,000×g for 5 minutes and the suspension buffer was removed. The remaining pellet was dissolved in citrate-phosphate buffer to remove the alumina coating and assess remaining protein. The suspension buffer was analyzed for IR-ovalbumin by measuring absorbance at 672 nm. Samples containing phosphate in the suspension buffer released IR-OVA from the particles into the suspension buffer at all time points and all temperatures. In contrast, samples containing histidine showed no detectable release regardless of incubation time or temperature. The trehalose-histidine suspension buffer was observed to prevent release of antigen into the suspension buffer during up to 8 weeks of incubation in the histidine-containing formulation.

[0089] Figure 4 presents plots of the release of IR-labeled ovalbumin after 1, 2, 4, or 8 weeks of incubation at room temperature or 50° C. Coated particles were suspended in trehalose-histidine or trehalose-phosphate buffer prior to incubation.

[0090] Example 5 In another exemplary experiment, spray-dried microparticles containing a protein antigen embedded in a glassy disaccharide matrix were coated with 100 layers of metal oxide (e.g., alumina) by atomic layer deposition (ALD). When suspended in aqueous solutions of phosphate, citrate, or sulfate, the ALD-coated microparticles were observed to rapidly release protein from the microparticle into the suspension medium. The data points for each buffer indicate the fraction of protein originally contained within the microparticle that was released into the suspension medium at each time point, while the lines represent an empirical fit to a dissolution model. Protein concentrations were measured using an o-phthalaldehyde fluorescence assay (OPA). Protein release from the microparticles is more rapid in solutions containing sulfate and citrate than in solutions containing potassium phosphate. For each of the suspensions tested, protein release from the microparticles was faster at higher salt concentrations. This is a counterexample to the claimed invention, for example, with respect to using histidine-containing formulations. This data demonstrates that ALD-coated microparticles are unstable in solutions of commonly used multivalent salts. For comparison, see FIG. 2B described herein, where the addition of histidine prevented the release of proteins from the microparticles.

[0091] Example 6 In another exemplary method, a powder containing a protein antigen labeled with an IR-active dye and embedded in a glassy trehalose-based matrix was prepared by spray drying. The powder was then coated with 500 layers of alumina using ALD. The coated powder was suspended in an aqueous solution containing a chelating agent (e.g., 20 mM EDTA), and the release of the IR dye-labeled protein antigen from the microparticles was monitored over a period of 4 days using infrared spectroscopy. (See, e.g., Figure 6).

[0092] FIG. 6 shows a graph of the percent release of antigen from coated microparticles suspended in a suspension buffer containing a chelating agent over several days of incubation in certain embodiments disclosed herein.

[0093] FIG. 7 represents an exemplary image of coating essentially dry microparticles containing at least one antigen. In this example, spray drying produces heat-stable particles containing APIs (antigens and adjuvants). Atomic layer deposition (ALD) applies a covalently bonded molecule-thick layer of metal oxide to the surface of the particle. The ALD coating (Al2O3) is insoluble in water and dissolves slowly in vivo. Multiple ALD cycles allow the coating thickness to be varied as desired. This layering allows for sustained or delayed release of antigens / adjuvants to subjects delivered with such formulations.

[0094] FIG. 8 shows an exemplary process represented by a schematic demonstrating metal oxide coating of an antigen showing first and second coatings of the antigen using metal oxide layer formation by an ALD process.

[0095] 9A-9C show schematic diagrams of certain embodiments disclosed herein of an antigen coated by metal oxide layer formation (9A), a second, different antigen added to the outer layer of a partially coated particle (9B), and a second coating of the same or different antigen (9C). These figures show prime dosing, prime-boost dosing, and multiple antigen dosing using the coated microparticles described herein.

[0096] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of this disclosure. Although the compositions and methods are described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes can be made in the compositions and methods described herein, and in the steps or sequence of steps of the methods, without departing from the concept, spirit and scope of the invention. More specifically, it is apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein, while still achieving the same or similar results. All such similar substitutions and modifications that are apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention, as defined by the appended claims.

Claims

1. 1. An aqueous composition comprising:

1. A composition comprising: microparticles containing one or more antigens coated with at least one metal oxide layer by atomic layer deposition (ALD); and a formulation comprising one or more non-chelating agents, wherein the formulation is an aqueous formulation.

2. 10. The composition of claim 1, wherein the one or more non-chelating agents comprise one or more of histidine, imidazole, glycine, bistrismethane, tris, bicine, or glycylglycine, or combinations thereof.

3. 10. The composition of claim 1, wherein the one or more non-chelating agents comprises at least one of histidine and imidazole.

4. The composition of any one of claims 1 to 3, wherein the concentration of the non-chelating agent is from about 0.1 mM to about 100.0 mM.

5. 10. The composition of claim 1, wherein the ALD coated one or more antigens comprise one or more metal oxide layers comprising one or more of aluminum oxide, aluminum alkoxide (e.g., alucone), silicon dioxide (SiO), titanium dioxide (TiO), silicon nitride (SiN), zinc oxide (ZnO), zircon (MLD), zirconia, hafnium oxide, either alone or in suitable combination compositions.

6. 10. The composition of claim 1, wherein the ALD coating comprises at least one covalently bonded molecular-thick layer of the metal oxide covering the one or more antigens.

7. The composition of any one of claims 1 to 3, further comprising one or more of a preservative, a surfactant or an antimicrobial agent.

8. 8. The composition of claim 7, wherein the one or more preservatives include at least one of benzyl alcohol, methylparaben, paraben, chlorobutanol, phenol, sorbic acid, cresol, meta-cresol, and other preservatives.

9. The composition of claim 1 , wherein the composition does not contain a chelating agent.

10. The composition according to any one of claims 1 to 3, wherein the composition does not contain at least one of sulfates and citrates.

11. 4. The composition of any one of claims 1 to 3, wherein the one or more antigens of the one or more antigens coated with the metal oxide layer comprise one or more of a polypeptide, a polynucleotide, a hybrid molecule of a polypeptide and a polynucleotide, a hybrid molecule of a polynucleotide, a microorganism, a virus, a virus-like particle, a bacterium, a bacteriophage, a fungus, a polysaccharide, a toxin, or a fragment thereof, or a small molecule.

12. The composition of claim 1 , wherein the formulation containing a non-chelating agent is isotonic.

13. The composition of any one of claims 1 to 3, wherein the pH of the composition is from about pH 6.0 to about pH 8.

0.

14. 4. The composition of any one of claims 1 to 3, wherein the microparticles comprising one or more antigens coated with at least one layer of metal oxide were essentially dry immediately prior to combining with the aqueous formulation.

15. 1. A method for increasing the stability of suspended antigen-containing ALD metal oxide-coated microparticles, the method comprising: introducing an aqueous formulation to essentially dry antigen-containing metal oxide-coated microparticles, the aqueous formulation comprising one or more non-chelating agents; and reducing the release of antigen from the antigen-containing metal oxide-coated microparticles.

16. 16. The method of claim 15, wherein the one or more non-chelating agents comprise one or more of histidine, imidazole, glycine, bistrismethane, tris, bicine, or glycylglycine, or combinations thereof.

17. 17. The method of claim 15 or 16, wherein the aqueous formulation further comprises one or more preservatives.

18. 17. The method of claim 15 or 16, wherein the concentration of the non-chelating agent in the aqueous formulation is comprised between 0.1 mM and 100 mM.

19. 17. The method of claim 15 or 16, wherein the pH of the aqueous formulation comprises a pH of about 6.0 to about 8.

0.

20. 17. The method of claim 15 or 16, wherein the antigen-containing metal oxide coated microparticles comprise antigen-containing ALD coated microparticles comprising one or more metal oxide coating layers comprising one or more of aluminum oxide, aluminum alkoxides (e.g., alucone), silicon dioxide (SiO2), titanium dioxide (TiO2), silicon nitride (Si3N4), zinc oxide (ZnO), zircon (MLD), zirconia, hafnium oxide, alone or in suitable combination compositions.

21. 16. The method of claim 15, wherein the one or more antigens comprise one or more of a polypeptide, a polynucleotide, a hybrid molecule of a polypeptide and a polynucleotide, a hybrid molecule of a polynucleotide, a microorganism, a virus, a virus-like particle, a bacterium, a bacteriophage, a fungus, a polysaccharide, a toxin, or a fragment thereof, or a small molecule.

22. A kit comprising the aqueous composition of any one of claims 1 to 3 and at least one container.

23. The kit of claim 22, wherein the aqueous composition comprises a multi-dose formulation.

24. 23. The kit of claim 22, wherein the aqueous composition is contained in one or more ready-to-administer delivery devices.

25. 25. The kit of claim 24, wherein the one or more ready-to-administer delivery devices comprise one or more syringes.

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