Formation of biologics and other agents via solvent removal

Abstract

The present disclosure generally relates to compositions comprising biologies and / or proteins. Some of the compositions described herein may be in the form of solid particles. In certain embodiments, the composition comprises a precipitant (e.g., interspersed with the biologic and / or protein). The composition may also comprise a dehydrating fluid (e.g., a relatively small amount of a dehydrating fluid). In some embodiments, suspensions of solid and / or substantially solid particles containing a protein and / or biologic and a precipitant are provided. For example, some suspensions described herein contain a plurality of solid particles containing a biologic and / or protein and a precipitant (e.g., a solid particle that is and / or comprises a composition described herein) suspended in an aqueous medium. Methods of forming such solid particles and other particles containing proteins and / or biologies are also disclosed.

Description

[0001] FORMATION OF BIOLOGICS AND OTHER AGENTS VIA SOLVENT REMOVAL

[0002] RELATED APPLICATIONS

[0003] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 715,472, filed November 1, 2024, and entitled “Formation of Biologies and Other Agents via Solvent Removal,” which is incorporated herein by reference in its entirety for all purposes.

[0004] TECHNICAL FIELD

[0005] Compositions comprising biologies and other agents and methods for making and using the same are generally described.

[0006] SUMMARY

[0007] Compositions comprising biologies and other agents and methods for making (e.g., via solvent removal) and using the same are generally described. The subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and / or a plurality of different uses of one or more systems and / or articles.

[0008] In one aspect, a composition is provided. In some embodiments, the composition comprises: a protein and / or biologic; a precipitant; and greater than or equal to 50 ppb and less than or equal to 25 mg / mL of a dehydrating fluid.

[0009] In another aspect, a suspension is provided. In some embodiments, the suspension comprises: an aqueous medium; a stabilizing agent contained within the aqueous medium; a plurality of solid particles suspended within the aqueous medium, each particle within the plurality of particles comprising: a protein and / or biologic, a precipitant, and a dehydrating fluid.

[0010] In another aspect, a method is provided. In some embodiments, the method comprises: providing a protein and / or biologic and a precipitant dissolved and / or suspended in a carrier fluid; removing at least some of the carrier fluid to a dehydrating phase; and forming a solid particle comprising the protein and / or biologic.

[0011] In another aspect, a method is provided. In some embodiments, the method comprises: providing a protein and / or biologic and a precipitant dissolved and / or suspended in a carrier fluid; in a droplet-forming phase, forming a droplet comprising the carrier fluid, the protein and / or biologic, and the precipitant; removing at least some of the carrier fluid of the droplet to a dehydrating phase; and forming a solid particle comprising the protein and / or biologic. Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the disclosure when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control.

[0012] BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Non-limiting embodiments of the present disclosure will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale unless otherwise indicated. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the disclosure shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure. In the figures:

[0014] FIG. 1A is a schematic diagram of a composition comprising a protein and / or a biologic and a precipitant, according to certain embodiments.

[0015] FIG. IB is a schematic diagram of a composition comprising a protein and / or a biologic, a precipitant, and a crosslinked polymer, according to certain embodiments.

[0016] FIG. 2A is a schematic diagram of a suspension comprising an aqueous medium containing a plurality of solid particles, according to some embodiments.

[0017] FIG. 2B is a schematic diagram of a suspension comprising an aqueous medium containing a stabilizing agent and a plurality of solid particles, according to some embodiments.

[0018] FIG. 2C is a schematic diagram of a suspension comprising an aqueous medium containing a stabilizing agent, precipitated biologic and / or protein, and a plurality of solid particles, according to some embodiments.

[0019] FIG. 3 is a schematic diagram of the formation of a solid particle comprising a biologic and / or protein and a precipitant, according to some embodiments.

[0020] FIG. 4A is a schematic diagram of the formation of a solid particle comprising a biologic and / or protein, a precipitant, and a cross-linked polymer, according to some embodiments.

[0021] FIG. 4B is a schematic diagram of the formation of a solid particle comprising a biologic and / or protein, a precipitant, and a cross-linked polymer, according to some embodiments.

[0022] FIG. 5A shows a schematic overview of a previous process for formulating high- concentration solid antibodies in aqueous suspension, in which poly(ethylene) glycol (PEG, a precipitant) is added to antibody solution, inducing precipitation of the antibody into an amorphous solid dispersion (ASD). The ASD is then concentrated and encapsulated into particles via centrifugal dripping, both steps being limited by ASD viscosities at higher concentrations (>300 mg / mL).

[0023] FIG. 5B shows a schematic overview of Microglassification™, a solvent-based dehydration process for making solid protein microbeads, in which a droplet of antibody solution is emulsified in an organic phase, extracting water from the droplet and causing precipitation of the antibody into its solid form. These highly concentrated microbeads can then be resuspended in a non-aqueous phase for delivery or reconstitution into a low concentration solution.

[0024] FIG. 5C shows a microparticle ASD formation process in which a droplet of antibody solution with precipitant (PEG) is emulsified in an organic phase, extracting water from the droplet and concentrating both antibody and PEG, which induces precipitation of the antibody into an ASD. The ASD is stabilized by PEG and the resulting microparticles can be resuspended at high concentrations in an aqueous phase with PEG for delivery.

[0025] FIG. 6A shows a phase diagram for IgG with varying PEG and IgG concentration, with discrete points correlating to experimental conditions and shaded areas corresponding to the estimated phase boundaries. Three phases are depicted, solution (solubilized IgG), LLPS (liquidliquid phase separation), and ASD (amorphous solid dispersion).

[0026] FIG. 6B shows time-lapse images of 60 mg / mL IgG, 2% PEG in pentanol (0.4% w / v Tween 80). Scale bar = 200 micrometers.

[0027] FIGs. 6C-6F show changes in scaled radius over time (min) for microparticles with varying concentrations of (FIG. 6C) IgG and (FIG. 6D) PEG in pentanol (0.4% w / v Tween80). The kinetic data for radius is plotted with the Epstein-Plesset model (solid grey line) for a pure water droplet of a comparable size, for reference. Final IgG concentrations (mg / mL), estimated from the initial droplet IgG concentration and the calculated concentration factor of the droplets, shown for varying (FIG. 6E) initial droplet IgG concentration (mg / mL), and (FIG. 6F) initial droplet PEG concentration (% w / v), where n=3 for each condition.

[0028] FIG. 7A shows an ASD-laden hydrogel microparticle formation process. A droplet of antibody solution with precipitant (PEG) and polymer (alginate) is emulsified in an organic outer phase, extracting water from the droplet and concentrating both the antibody and PEG, which induces precipitation of the antibody into an ASD. Cross-linker (calcium) in the organic phase cross-links alginate simultaneously to dehydration and precipitation, resulting in a highly concentrated ASD-laden hydrogel microparticle. The ASD is stabilized by PEG and the resulting microparticles can be resuspended in an aqueous phase with PEG for delivery, with hydrogel encapsulation improving the stability and injectability of the formulation.

[0029] FIG. 7B shows images of individual ASD-laden alginate microparticles demonstrating resuspension of the particles in PEG solution after dehydration, and dissolution of the ASD after release in simulated body fluid.

[0030] FIG. 7C shows the change in scaled radius over time (min) for hydrogel microparticles with various calcium concentration in pentanol (0.4% w / v Tween80). The kinetic data for radius is plotted with the Epstein-Plesset model (solid grey line) for a pure water droplet of a comparable size, for reference.

[0031] FIG. 8A shows a schematic of the microfluidic cross-junction used for microparticle generation. The continuous phase, pentanol, is fed into two inlets perpendicular to the antibody solution, causing pinch-off of the dispersed (antibody) phase, where the antibody droplets are dried in the continuous stream and fed into a reservoir for particle collection.

[0032] FIG. 8B shows mean particle diameter (micrometers) versus flow rate ratio for the microparticle generation process.

[0033] FIG. 8C shows representative images of ASD-laden hydrogel particles produced at various flow rate ratios in the microfluidic process. Scale bar = 200 micrometers.

[0034] FIG. 8D shows the apparent solubility of IgG (mg / mE) from the hydrogel particles in aqueous solutions of varying PEG concentration (% w / v), based on a total concentration of 1 mg / mL IgG.

[0035] FIG. 9A is a digital camera image of a syringe loaded with the microparticle formulation at a formulation concentration of 360+9 mg / mL.

[0036] FIG. 9B shows the injection force (N) versus plunger distance traveled (mm) for an IgG solution control (blue) and the microparticle formulation (orange) (n=3). Maximum injection force is shown for reference.

[0037] FIG. 9C shows the mean residual ellipticity (deg cm2 dmol-1) from circular dichroism measurements of native IgG reference and IgG reconstituted from hydrogel microparticles.

[0038] FIG. 9D shows the secondary structure composition as calculated from FTIR measurements for native IgG reference and IgG reconstituted from hydrogel microparticles.

[0039] FIG. 9E shows the release profile over time for IgG released from hydrogel microparticles in simulated body fluid (SBF) at 37 °C. Time-lapse images of an ASD-laden hydrogel microparticle in SBF are inset (scale bar = 50 micrometers).

[0040] FIG. 10 shows a schematic of amorphous solid antibodies being encapsulated into hydrogel microparticles through a solvent-based dehydration process, in which the solvent extracts water from antibody solution droplets, causing the antibody to concentrate, and precipitation of the antibody into amorphous solid is induced, and the particles are then resuspended into aqueous solution in which they are injectable for subcutaneous delivery at high concentrations.

[0041] FIGs. 11A-11B shows log S (solubility, mg / mL) for IgG and PEG mixtures with various initial conditions, based off the data in Figure 2a. Solubility data points are differentiated by (FIG. 11 A) initial IgG concentration and (FIG. 1 IB) resulting phase transition

[0042] FIGs. 12A-12B show brightfield microscopy images and digital camera images after centrifugation of IgG-PEG mixtures, characterized as (FIG. 12 A) liquid- liquid phase separation (EEPS) and (FIG. 12B) amorphous solid dispersion (ASD).

[0043] FIG. 13 shows scaled radius versus scaled time for the dehydration of pure water droplets in pentanol, for droplets with varying initial radii (n=5). The data were fitted to the Epstein- Plesset model to extrapolate the fitted diffusion coefficient for each condition (Dfit). R-squared values are shown for each experiment. The average of these Dfit were taken as D for further experiments. All control experiments were plotted onto a master curve.

[0044] FIGs 14A shows time-lapse images of dehydration process for a droplet (60 mg / mL IgG, PEG 2% w / v), no surfactant (0% w / v Tween 80) in pentanol.

[0045] FIG. 14B shows time-lapse images of dehydration process for a droplet without PEG (60 mg / mL IgG, 0% w / v PEG) in pentanol (0.4% w / v Tween 80). Scale bar = 200 micrometers.

[0046] FIG. 15 shows images of individual ASD microparticles, without alginate, after dehydration in pentanol (top) and resuspension in 15% w / v PEG solution (bottom). Subsequent dissolution of the microparticles in simulated body fluid (SBF) was not shown due to the rapid dispersion and dissolution of the ASD in SBF. Scale bar = 100 micrometers.

[0047] FIG. 16 shows final IgG concentrations (mg / mL), estimated from the initial droplet IgG concentration (60 mg / mL) and the calculated concentration factor of the droplets (n=3 for each condition), shown for varying pentanol Ca2+concentration (% w / v).

[0048] FIG. 17 shows representative second-derivative FTIR-ATR spectra for native IgG reference (blue) and reconstituted IgG from hydrogel particles (orange).

[0049] FIGs. 18A-18B show UV traces from size exclusion chromatography of released IgG from (FIG. 18 A) native control sample and (FIG. 18B) alginate microparticles.

[0050] FIG. 19 is a schematic of a process to produce microparticles containing a high concentration of amorphous solid forms of proteins or other biologies.

[0051] FIGs. 20A-20B show high-concentration precipitation of proteins via solvent-based dehydration with PEG. FIG. 20A shows a solution of 40 mg / mL IgG and 4% PEG, before (top) and after (bottom) dehydration. FIG. 20B shows a solution of 40 mg / mL IgG and 0% PEG, before (top) and after (bottom) dehydration.

[0052] FIGs. 21A-21B show high-concentration precipitation in the presence of PEG and another polymer. FIG. 21A shows a solution of 10 mg / mL IgG, 2% PEG and 0.2% alginate, before (top) and after (bottom) dehydration. FIG. 2 IB shows volume reduction of a microparticle containing 40 mg / mL IgG; 1% w / v PEG; and 0.2% w / v alginate in the initial droplet. Pictures are shown chronologically from top-bottom.

[0053] FIGs. 22A-22B show co-formulation of 2 different proteins into the same microparticle. FIG. 22A shows an initial solution of 40 mg / mL IgG and 4% PEG, before (top) and after (bottom) dehydration. FIG. 22B shows an initial solution of 20 mg / mL IgG, 20 mg / mL BSA, and 4% PEG, before (top) and after (bottom) dehydration.

[0054] FIGs. 23A-23B show formed protein particles can be resuspended in an aqueous solution containing PEG and do not dissolve. FIG. 23A shows an image of IgG (40 mg / mL, 4% PEG) droplets emulsified and then dehydrated in 1 -pentanol. FIG. 23B shows an image of IgG particles resuspended in 25% PEG solution after washing with ethanol.

[0055] DETAILED DESCRIPTION

[0056] The present disclosure generally relates to compositions comprising biologies and / or proteins. Some of the compositions described herein may be in the form of solid particles. In certain embodiments, the composition comprises a precipitant (e.g., interspersed with the biologic and / or protein). The composition may also comprise a dehydrating fluid (e.g., a relatively small amount of a dehydrating fluid). In some embodiments, suspensions of solid and / or substantially solid particles containing a protein and / or biologic and a precipitant are provided. For example, some suspensions described herein contain a plurality of solid particles containing a biologic and / or protein and a precipitant (e.g., a solid particle that is and / or comprises a composition described herein) suspended in an aqueous medium. Methods of forming such solid particles and other particles containing proteins and / or biologies are also disclosed.

[0057] Many therapeutic biologies and / or proteins (e.g., therapeutic monoclonal antibodies (mAbs)) are presently used for the treatment of diseases such as cancers, autoimmune diseases, and other chronic and acute illnesses, and the development of therapeutic biologies and / or proteins for use in treating other diseases is an active area of research. Therapeutic biologies and / or proteins are typically administered to patients by intravenous infusion, which can be costly, time-consuming, and can negatively impact patient compliance with treatment protocols. - 1 -

[0058] It would be advantageous to administer such therapeutic biologies and / or proteins via subcutaneous injection, since subcutaneous injections can be self-administered by patients, which generally reduces costs and improves patient compliance and outcomes. However, the injection volume for subcutaneous delivery is limited (e.g., to approximately less than or equal to 2 mL), thus necessitating highly concentrated formulations (e.g., approximately 200 mg / mL of the biologic and / or protein) to meet the dosing requirements for therapies using biologies and / or proteins, such as mAb therapies. High-concentration solutions of proteins and / or biologies often have prohibitively high viscosities due to the intermolecular interactions of the proteins and / or biologies, which can render such solutions difficult, dangerous, or uncomfortable to inject subcutaneously, and lead to higher likelihood of injection site irritation. Accordingly, compositions comprising biologies and / or proteins that allow for the formation of highly concentrated formulations with relatively low viscosities are desirable.

[0059] The compositions described herein may take the form of solid or substantially solid particles comprising a protein and / or biologic and a precipitant. These compositions may contain high concentrations of the biologic and / or protein. Advantageously, the compositions described herein may be able to be suspended in high concentrations in an aqueous medium, unlike similar compositions that do not contain a precipitant (e.g., solid particles such as amorphous dispersions containing a protein and / or biologic without a precipitant), which often dissolve when suspended in high concentrations in an aqueous solution, thus forming a protein and / or biologic solution having a high viscosity as described above. It is also advantageous for the compositions described herein to be able to be suspended in high concentrations in an aqueous medium (e.g., an aqueous solution comprising a stabilizing agent), rather than only being able to be suspended in high concentrations in an organic medium, as is the case for some existing solid compositions comprising biologies and / or proteins. As described in greater detail below, aqueous solutions are preferred for injectable formulations over non-aqueous (e.g., organic) solutions, as they are less likely to cause injection site irritation or other negative patient outcomes, and many stabilizing agents used in aqueous solutions are already well-known pharmaceutical excipients with established safety records.

[0060] As noted above, the compositions described herein may comprise a biologic and / or a protein. As used herein, “biologic” is given its ordinary meaning in the art, and refers to a product produced by and / or isolated from a living organism. As used herein, a “protein” may refer to any protein, protein fragment, or peptide chain. The composition may be provided in any of a variety of suitable forms, such as a bulk composition or a particle (e.g., as shown in FIG. 1A as composition 100a). Composition 100a shown in FIG. 1A comprises biologic and / or protein 101. The biologic and / or protein may be any of a variety of suitable biologies and / or proteins of interest or use for a variety of applications (e.g., therapeutic and / or research applications). For example, the composition may comprise a therapeutic biologic and / or protein (e.g., the biologic and / or protein may be an active pharmaceutical ingredient). In some embodiments, the protein and / or biologic may comprise a monoclonal antibody, a peptide, a bispecific antibody, a nucleotide, an oligonucleotide, a recombinant protein, and / or an enzyme.

[0061] The compositions described herein may comprise a precipitant. As used herein, “precipitant” means a reagent that causes or facilitates the precipitation of a solid composition from a solution and / or suspension. The precipitant may be mixed with (e.g., interspersed with) the biologic and / or protein within the composition. A precipitant that is interspersed with the biologic and / or protein may form a substantially homogeneous mixture of the precipitant with the biologic and / or protein. For example, the composition 100a shown in FIG. 1A comprises precipitant 102 interspersed with biologic and / or protein 101. In some embodiments, the presence of the precipitant may advantageously increase the stability of the composition (e.g., in the form of a solid particle), particularly when the composition is suspended in an aqueous solution, as described in greater detail elsewhere herein. Any of a variety of suitable precipitants may be used. For example, the precipitant may comprise polyethylene glycol, polyethyleneimine, ammonium sulfate, ethanol, acetone, and / or trichloroacetic acid. Suitable precipitants may be selected in combination with a biologic and / or protein such that the precipitant facilitates the precipitation of the biologic and / or protein in combination with the precipitant from an aqueous solution and / or suspension at a particular concentration thereof, as described in greater detail elsewhere herein.

[0062] The precipitant may be present in the composition in any of a variety of suitable amounts. For example, the precipitant may be present in the composition an amount greater than or equal to 0.5 wt%, greater than or equal to 1 wt%, greater than or equal to 2 wt%, greater than or equal to 3 wt%, greater than or equal to 4 wt%, greater than or equal to 5 wt%, greater than or equal to 7 wt%, greater than or equal to 10 wt%, greater than or equal to 12 wt%, greater than or equal to 15 wt%, greater than or equal to 20 wt%, greater than or equal to 25 wt%, or greater. In some embodiments, the precipitant is present in the composition in an amount less than or equal to 30 wt%, less than or equal to 25 wt%, less than or equal to 20 wt%, less than or equal to 15 wt%, less than or equal to 12 wt%, less than or equal to 10 wt%, less than or equal to 7 wt%, less than or equal to 5 wt%, less than or equal to 4 wt%, less than or equal to 3 wt%, less than or equal to 2 wt%, less than or equal to 1 wt%, or less. Combinations of these ranges are also possible. For example, the precipitant may be present in the composition in an amount of greater than or equal to 0.5 wt% and less than or equal to 30 wt%, or greater than or equal to 1 wt% and less than or equal to 25 wt%. Other ranges are also possible.

[0063] In some embodiments, the composition comprises a dehydrating fluid. As used herein, a “dehydrating fluid” is a fluid into which water may diffuse and / or be absorbed from an adjacent phase, as described in greater detail below. The composition may comprise the dehydrating fluid in a relatively small amount. In some instances, the dehydrating fluid may be introduced to the composition during a manufacturing process (e.g., as described in greater detail elsewhere herein). For example, the composition may comprise the dehydrating fluid in an amount less than or equal to 25 mg / mL, less than or equal to 15 mg / mL, less than or equal to 10 mg / mL, less than or equal to 5 mg / mL, less than or equal to 1 mg / mL, less than or equal to 0.5 mg / mL, less than or equal to 0.1 mg / mL, less than or equal to 0.05 mg / mL, less than or equal to 0.005 mg / mL, less than or equal to 0.0005 mg / mL, or less. The composition may comprise dehydrating fluid in an amount greater than or equal to 0 mg / mL, greater than or equal to 0.00005 mg / mL, greater than or equal to 0.0005 mg / mL, greater than or equal to 0.005 mg / mL, greater than or equal to 0.05 mg / mL, greater than or equal to 0.1 mg / mL, greater than or equal to 0.5 mg / mL, greater than or equal to 1 mg / mL, greater than or equal to 5 mg / mL, greater than or equal to 10 mg / mL, greater than or equal to 15 mg / mL, or greater. Combinations of these ranges are also possible. For example, the composition may comprise the dehydrating fluid in an amount greater than or equal to 0 mg / mL and less than or equal to 25 mg / mL, greater than or equal to 0.00005 mg / mL and less than or equal to 25 mg / mL, or greater than or equal to 0.0005 mg / mL and less than or equal to 25 mg / mL. As used herein, a composition comprising less than or equal to 25 mg / mL indicates that the composition comprises less than or equal to 25 mg of the dehydrating fluid per mL of the composition. In some embodiments, the composition is substantially free of dehydrating fluid (e.g., may comprise no dehydrating fluid, or may comprise only a trace amount of the dehydrating fluid).

[0064] The dehydrating fluid may comprise any of a variety of suitable fluids. For example, the dehydrating fluid may comprise a class 3 ICH solvent. In some embodiments, the dehydrating fluid may comprise one or more alcohols, such as a propanol, a butanol, 2-methyl-l-proponal; 2- methyl-2-propanol, tert-butanol, a pentanol such as 1-pentanol, 3-methyl-l -butanol, 2,2- dimethyl-1 -propanol, and / or cyclopentanol, a hexanol, cyclohexanol, a heptanol, an octanol, a nonanol, a decanol, 2-propen-l-ol; benzyl alcohol, phenylmethanol, diphenylmethanol, undecanol, a dodecanol, a propyldecanol, a butadecanol, a pentadecanol, a hexadecanol; and / or triphenylmethanol. In some embodiments, the dehydration composition comprises an isomer of a linear alcohol (e.g., 2-octanol, 3-pentanol, 4-decanol), a derivative of a linear alcohol or their isomer (e.g., octyldodecanol, neopentyl alcohol), a di-, tr-, or quad-hydroxylated material (e.g., 1,4-butanediol, glycerin), an unsaturated alcohol (e.g., a cyclic, olefinic or alkynyl alcohol such as e.g., cyclohexanol, geraniol, oleic alcohol), and / or an alcohol incorporating an internal and / or external heteroatoms (e.g., polyethylene glycols, polypropylene glycols, lactates, etc.).

[0065] In some embodiments, the composition comprises a crosslinked polymer. As used herein, “crosslinked polymer” means a polymer that is at least partially crosslinked, at least to the extent that the polymer can function as described as follows. The polymer can be crosslinked to any extent, as would be understood and can be accomplished by those of ordinary skill in the art, with the extent of crosslinking tested and selected for optimal performance as described. The crosslinked polymer may advantageously form a scaffold to support and / or stabilize the biologic and / or protein within the composition. For example, as shown in FIG. IB, composition 100b comprises crosslinked polymer 103 forming a scaffold to support and / or stabilize the biologic and / or protein 101. Any of a variety of suitable crosslinked polymers may be used. For example, the crosslinked polymer may comprise a crosslinked polyalkylene oxide, a crosslinked vinylsulfuone / thiol, crosslinked PEG, crosslinked PEGDA, a natural polysaccharide, a modified polysaccharide, and / or crosslinked alginate. Such a composition may also comprise a precipitant, as shown in FIG. IB as precipitant 102.

[0066] The composition may comprise a crosslinked polymer in any of a variety of suitable amounts. For example, the composition may comprise the crosslinked polymer in an amount of greater than or equal to 0.01 wt%, greater than or equal to 0.05 wt%, greater than or equal to 0.1 wt%, greater than or equal to 0.5 wt%, greater than or equal to 1 wt%, greater than or equal to 2 wt%, greater than or equal to 3 wt%, greater than or equal to 4 wt%, or greater. In some embodiments, the composition comprises the crosslinked polymer in an amount of less than or equal to 5 wt%, less than or equal to 4 wt%, less than or equal to 3 wt%, less than or equal to 2 wt%, less than or equal to 1 wt %, less than or equal to 0.5 wt%, less than or equal to 0.1 wt%, less than or equal to 0.05 wt%, or less. Combinations of these ranges are also possible. For example, the composition may comprise a crosslinked polymer in an amount of greater than or equal to 0.01 wt% and less than or equal to 5 wt%, or greater than or equal to 0.05 wt% and less than or equal to 4 wt%. Other ranges are also possible.

[0067] In some embodiments, the composition comprises a surfactant. The presence of a surfactant in the composition may advantageously prevent and / or reduce the risk of particle agglomeration (e.g., in a suspension, as described below). The surfactant may also have a variety of advantages related to particle morphology, such as maintaining a smooth particle surface morphology of the composition. The composition may comprise any of a variety of suitable surfactants. For example, the surfactant may comprise a polysorbate, a span, a pegylated fatty ester, a pegylated fatty ether, a sucrose ester, a block copolymer, and / or an ethoxylated triglyceride.

[0068] The surfactant may be present in the composition in any of a variety of suitable amounts. For example, the surfactant may be present in the composition in an amount of greater than or equal to 0.01 wt%, greater than or equal to 0.05 wt%, greater than or equal to 0.1 wt%, greater than or equal to 0.25 wt%, greater than or equal to 0.5 wt%, greater than or equal to 0.75 wt%, or greater. In some embodiments, the surfactant is present in the composition in an amount of less than or equal to 1 wt%, less than or equal to 0.75 wt%, less than or equal to 0.5 wt%, less than or equal to 0.25 wt%, less than or equal to 0.1 wt%, less than or equal to 0.05 wt%, or less. Combinations of these ranges are also possible. For example, the surfactant may be present in an amount of greater than or equal to 0.01 wt% and less than or equal to 1 wt%, or greater than or equal to 0.05 wt% and less than or equal to 1 wt%. Other ranges are also possible.

[0069] In some embodiments, the composition comprises a salt. The salt may be dissolved and / or interspersed within the composition. Any of a variety of suitable salts may be used, such as sodium chloride, potassium chloride, and / or calcium chloride. The salt may be present in the composition in any of a variety of suitable concentrations. For example, the salt may be present in the composition in an amount of greater than or equal to 5 mM, greater than or equal to 10 mM, greater than or equal to 25 mM, greater than or equal to 50 mM, greater than or equal to 75 mM, greater than or equal to 100 mM, greater than or equal to 125 mM , greater than or equal to 150 mM, greater than or equal to 175 mM, or greater In some embodiments, the salt is present in the concentration in an amount of less than or equal to 200 mM, less than or equal to 175 mM, less than or equal to 150 mM, less than or equal to 125 mM, less than or equal to 100 mM, less than or equal to 75 mM, less than or equal to 50 mM, less than or equal to 25 mM, less than or equal to 10 mM, or less. Combinations of these range are also possible. For example, the salt may be present in the compound in an amount of greater than or equal to 5 mM and less than or equal to 200 mM, or greater than or equal to 10 mM and less than or equal to 150 mM. Other ranges are also possible.

[0070] In some embodiments, a suspension is provided. The suspension may comprise an aqueous medium and a plurality of solid particles suspended therein (e.g., a plurality of solid particles comprising a protein and / or biologic, a precipitant, and optionally a dehydrating fluid). For example, suspension 200a shown in FIG. 2A comprises aqueous medium 240 and, contained therein, a plurality of solid particles 250 comprising protein and / or biologic 201 and precipitant 202. The plurality of solid particles contained within the suspension may have any of the properties, features, and / or characteristics of the compositions described above and shown in FIGS. 1A-1B. For example, each of the solid particles may comprise any of the biologies and / or proteins described above. Each of the solid particles may comprise a precipitant in any of the amounts and / or having any of the compositions described above. Each particle in the plurality of particles (e.g., all of the particles, a subset of the particles, etc.) may further comprise any one of or any combination of the other components of a composition described above, such as a surfactant, a polymer, and / or a dehydrating fluid. In some embodiments, each of the solid particles may comprise a dehydrating fluid of any of the types described above and / or in any of the amounts described above. For example, each of the solid particles may comprise a dehydrating fluid in an amount of greater than or equal to 0.00005 mg / mL, less than or equal to 25 mg / mL, or may contain no dehydrating fluid.

[0071] Each of the solid particles within the suspension may contain a surfactant. For example, the solid particles may contain a surfactant having any of the compositions and / or in any of the amounts described above for the surfactant contained within the composition. Similarly, each of the solid particles within the suspension may contain a crosslinked polymer. For example, the solid particles may contain a crosslinked polymer having any of the compositions and / or in any of the amounts described above for the crosslinked polymer contained within the composition.

[0072] The suspension may comprise solid particles suspended in the aqueous medium in any of a variety of suitable concentrations. In some embodiments, the solid particles may be present in a relatively high concentration. For example, the suspension may comprise solid particles suspended in the aqueous medium in a concentration of greater than or equal to 350 mg / mL, greater than or equal to 375 mg / mL, greater than or equal to 400 mg / mL, greater than or equal to 425 mg / mL, greater than or equal to 450 mg / mL, or greater. In some embodiments, the suspension comprises solid particles suspended in the aqueous medium in a concentration of less than or equal to 500 mg / mL, less than or equal to 475 mg / mL, less than or equal to 450 mg / mL, less than or equal to 425 mg / mL, less than or equal to 400 mg / mL, less than or equal to 375 mg / mL, or less. Combinations of these ranges are also possible. For example, the suspension may comprise solid particles suspended in the aqueous medium in an amount of greater than or equal to 350 mg / mL and less than or equal to 500 mg / mL.

[0073] Advantageously, the suspension may have a relatively low viscosity and / or a relatively low injection glide force, even when the solid particles are present in a relatively high concentration. The injection glide force refers to the plateau regime of the injection force of the suspension through a 25-gauge needle through which the suspension is flowed at a rate of 25 microliters / second, and is a clinically relevant parameter for the viability of a subcutaneous injection, which would typically be administered using a 25-gauge needle. Additional details of injection force testing are described in Example 1 below. The injection glide force of a suspension described herein injected through a 25-gauge needle at a flow rate of 25 microliters / second may be less than or equal to 40 N, or preferably within a range of approximately 10 N - 20 N. An injection glide force of less than 40 N indicates that the suspension may be suitable for subcutaneous injection.

[0074] The aqueous medium may comprise an aqueous solution with any of a variety of suitable compositions. For example, the aqueous medium may comprise a buffer, such as a HEPES buffer, a MES buffer, a sodium acetate buffer, a sodium citrate buffer, an ammonium sulfate buffer, a phosphate buffer, and / or a potassium phosphate buffer.

[0075] In some embodiments, the aqueous medium of the suspension contains a stabilizing agent. As used herein, a “stabilizing agent” is given its ordinary meaning in the art and refers to a reagent that is configured to enhance the stability of a suspension (e.g., by preventing and / or reducing the risk of agglomeration of solid particles and / or preventing or reducing the risk of a solid particle precipitating out of the suspension). For example, as shown in FIG. 2B, suspension 200b comprises aqueous medium 240 comprising stabilizing agent 210, where solid particles 250 comprising protein and / or biologic 201 and precipitant 202 are suspended. It may be advantageous for the aqueous medium to contain a stabilizing agent, as the stabilizing agent may increase the stability (e.g., prevent and / or mitigate agglomeration) of the solid particles suspended in the aqueous phase (e.g., stabilize the biologic and / or protein contained therein). The aqueous medium of the suspension may comprise any of a variety of suitable stabilizing agents. For example, the stabilizing agent may comprise polyethylene glycol, polyethyleneimine, ammonium sulfate, ethanol, acetone, and / or trichloroacetic acid. In some embodiments, the stabilizing agent contained within the aqueous medium and the precipitant contained within the solid particles have the same composition. In some embodiments, the stabilizing agent contained within the aqueous medium and the precipitant contained within the solid particles have different compositions.

[0076] The stabilizing agent may be present in the aqueous medium in any of a variety of suitable amounts. For example, the stabilizing agent may be present in the aqueous medium in an amount of greater than or equal to 0 wt%, 0.5 wt%, greater than or equal to 1 wt%, greater than or equal to 2 wt%, greater than or equal to 3 wt%, greater than or equal to 4 wt%, greater than or equal to 5 wt%, greater than or equal to 7 wt%, greater than or equal to 10 wt%, greater than or equal to 12 wt%, greater than or equal to 15 wt%, greater than or equal to 20 wt%, greater than or equal to 25 wt%, or greater. In some embodiments, the precipitant is present in the aqueous medium in an amount less than or equal to 30 wt%, less than or equal to 25 wt%, less than or equal to 20 wt%, less than or equal to 15 wt%, less than or equal to 12 wt%, less than or equal to 10 wt%, less than or equal to 7 wt%, less than or equal to 5 wt%, less than or equal to 4 wt%, less than or equal to 3 wt%, less than or equal to 2 wt%, less than or equal to 1 wt%, or less. Combinations of these ranges are also possible. For example, the precipitant may be present in the aqueous medium in an amount of greater than or equal to 0.5 wt% and less than or equal to 15 wt%, or greater than or equal to 5 wt% and less than or equal to 30 wt%.

[0077] The aqueous medium may comprise precipitated biologic and / or protein contained therein. For example, as shown in FIG. 2C, suspension 200c comprises aqueous medium 240 comprising stabilizing agent 210 and precipitated biologic and / or protein 260, where solid particles 250 comprising protein and / or biologic 201 and precipitant 202 are suspended within aqueous medium 240. For example, the aqueous medium may comprise monomers of the biologic and / or protein. In some embodiments, greater than or equal to 90% of the precipitated biologic and / or protein contained within the aqueous medium may comprise monomers of the biologic and / or protein.

[0078] In some embodiments, the aqueous medium comprises a surfactant dissolved therein. The aqueous medium may comprise any of a variety of suitable surfactants. For example, the surfactant may comprise a polysorbate, a span, a pegylated fatty ester, a pegylated fatty ether, a sucrose ester, a block copolymer, and / or an ethoxylated triglyceride.

[0079] The surfactant may be present in the aqueous medium in any of a variety of suitable amounts. For example, the surfactant may be present in the aqueous medium in an amount of greater than or equal to 0.01 wt%, greater than or equal to 0.05 wt%, greater than or equal to 0.1 wt%, greater than or equal to 0.25 wt%, greater than or equal to 0.5 wt%, greater than or equal to 0.75 wt%, or greater. In some embodiments, the surfactant is present in the aqueous medium in an amount of less than or equal to 1 wt%, less than or equal to 0.75 wt%, less than or equal to 0.5 wt%, less than or equal to 0.25 wt%, less than or equal to 0.1 wt%, less than or equal to 0.05 wt%, or less. Combinations of these ranges are also possible. For example, the surfactant may be present in the aqueous medium an amount of greater than or equal to 0.01 wt% and less than or equal to 1 wt%, or greater than or equal to 0.05 wt% and less than or equal to 1 wt%. Other ranges are also possible.

[0080] In certain embodiments, a method is provided. The method may comprise providing a protein and / or a biologic and a precipitant dissolved and / or suspended in a carrier fluid. For example, as shown in FIG. 3, stage 1 of method 300 comprises providing protein and / or biologic 301 and precipitant 302 dissolved and / or suspended in carrier fluid 370. The protein and / or biologic and precipitant dissolved and / or suspended in the carrier fluid may be provided in any of a variety of suitable forms. For example, a bulk solution of the carrier fluid, biologic and / or protein, and precipitant may be provided. In some embodiments, a pre-formed droplet and / or particle (e.g., a microparticle) comprising the carrier fluid, the biologic and / or protein, and the precipitant may be provided. In some embodiments, the method may comprise forming a preformed droplet and / or particle by first providing a protein and / or biologic and a precipitant dissolved and / or suspended in a carrier fluid (e.g., as a bulk solution), and then, in a dropletforming phase, forming a droplet comprising the carrier fluid, the protein and / or biologic, and the precipitant.

[0081] The biologic and / or protein may be any biologic and / or protein described above and / or any other suitable biologic and / or protein. The carrier fluid may comprise any suitable fluid capable of dissolving the protein and / or biologic. For example, the carrier fluid may comprise an aqueous solution. The aqueous solution may comprise water and other components such as a precipitant, a salt, a polymer, and / or a surfactant, as described elsewhere herein. The aqueous solution may comprise a buffer, such as a HEPES buffer, a MES buffer, a sodium acetate buffer, a sodium citrate buffer, an ammonium sulfate buffer, a phosphate buffer, and / or a potassium phosphate buffer.

[0082] As noted above, the carrier fluid may comprise a precipitant dissolved and / or suspended therein. The precipitant may be any of the precipitants described above, such as polyethylene glycol, polyethyleneimine, ammonium sulfate, ethanol, acetone, and / or trichloroacetic acid. The precipitant may be present in a relatively low concentration in the carrier fluid. For example, the precipitant may be present in an amount of greater than or equal to 0.5 wt%, greater than or equal to 1 wt%, greater than or equal to 2 wt%, greater than or equal to 3 wt%, or greater than or equal to 4 wt%. The carrier fluid may comprise the precipitant in an amount of less than or equal to 5 wt%, less than or equal to 4 wt%, less than or equal to 3 wt%, or less than or equal to 2 wt%. Combinations of these ranges are also possible. For example, the carrier fluid may comprise the precipitant in an amount of greater than or equal to 0.5 wt% and less than or equal to 5 wt%.

[0083] In some embodiments, the method comprises removing at least some of the carrier fluid to a dehydrating phase (e.g., a dehydrating fluid). For example, the method may comprise placing the carrier fluid comprising dissolved and / or suspended protein and / or biologic and precipitant (e.g., a pre-formed droplet and / or particle containing the carrier fluid comprising dissolved and / or suspended protein and / or biologic and precipitant) in and / or adjacent a dehydrating phase. In some embodiments, droplets comprising the protein and / or biologic, the precipitant, and the carrier fluid may be formed within the dehydrating phase (e.g., from a bulk solution comprising the carrier fluid, biologic and / or protein, and precipitant). The droplets comprising the protein and / or biologic, precipitant, and carrier fluid may be formed within the dehydrating phase via any of a variety of known microfluidic droplet generation techniques, such as generation of droplets at a microfluidic T-junction into which the solution comprising the carrier fluid containing the protein and / or biologic and precipitant and the dehydrating fluid are simultaneously flowed.

[0084] When the carrier fluid comprising suspended and / or dissolved protein and / or biologic and precipitant is placed in and / or adjacent to the dehydrating phase (e.g., by the formation of a droplet within the dehydrating fluid and / or placement of a pre-formed droplet within the dehydrating fluid), at least some of the carrier fluid (e.g., at least some of the water of the carrier fluid, when the carrier fluid is an aqueous solution) may be removed to the dehydrating fluid via diffusion. For example, as shown in FIG. 3, in stage 2 of the method 300, water 345 from carrier fluid 340 is removed to dehydrating phase 380. In some embodiments in which a droplet comprising the carrier fluid, protein and / or biologic, and precipitant is formed within the dehydrating fluid, removing at least some of the carrier fluid to the dehydrating fluid may occur and / or begin to occur substantially simultaneously with the formation of the droplet.

[0085] The dehydrating phase may comprise any of a variety of suitable dehydrating fluids. For example, the dehydrating fluid may comprise a class 3 ICH solvent. In some embodiments, the dehydrating fluid may comprise one or more alcohols, such as a propanol, a butanol, 2-methyl-l- proponal; 2-methyl-2-propanol, tert-butanol, a pentanol such as 1 -pentanol, 3-methyl-l -butanol, 2,2-dimethyl-l -propanol, and / or cyclopentanol, a hexanol, cyclohexanol, a heptanol, an octanol, a nonanol, a decanol, 2-propen-l-ol; benzyl alcohol, phenylmethanol, diphenylmethanol, undecanol, a dodecanol, a propyldecanol, a butadecanol, a pentadecanol, a hexadecanol; and / or triphenylmethanol. In some embodiments, the dehydration composition comprises an isomer of a linear alcohol (e.g., 2-octanol, 3-pentanol, 4-decanol), a derivative of a linear alcohol or their isomer (e.g., octyldodecanol, neopentyl alcohol), a di-, tr-, or quad-hydroxylated material (e.g., 1,4-butanediol, glycerin), an unsaturated alcohol (e.g., a cyclic, olefinic or alkynyl alcohol such as e.g., cyclohexanol, geraniol, oleic alcohol), and / or an alcohol incorporating an internal and / or external heteroatoms (e.g., polyethylene glycols, polypropylene glycols, lactates, etc.).

[0086] The dehydrating phase may comprise any of a variety of suitable additives and / or other components contained within the dehydrating fluid. For example, in some embodiments, the dehydrating fluid comprises a surfactant, such as a polysorbate, a span, a pegylated fatty ester, a pegylated fatty ether, a sucrose ester, a block copolymer, and / or an ethoxylated triglyceride. In some embodiments, the surfactant present in the dehydrating fluid may advantageously modify and / or control the interfacial tension at an interface of the carrier fluid (e.g., the droplet and / or particle comprising the carrier fluid) and the dehydrating fluid, and may thus facilitate a droplet and / or particle comprising the carrier fluid, precipitant, and protein and / or biologic maintaining a substantially spherical shape when suspended in the dehydrating fluid.

[0087] The surfactant may be present in the dehydrating fluid in any of a variety of suitable amounts. For example, the surfactant may be present in the dehydrating fluid in an amount of greater than or equal to 0 wt%, greater than or equal to 0.1 wt%, greater than or equal to 0.2 wt%, greater than or equal to 0.3 wt%, greater than or equal to 0.4 wt%, greater than or equal to 0.5 wt%, greater than or equal to 0.6 wt%, greater than or equal to 0.7 wt%, greater than or equal to 0.8 wt%, greater than or equal to 0.9 wt%, or greater. In some embodiments, the surfactant is present in the dehydrating fluid an amount of less than or equal to 1 wt%, less than or equal to 0.9 wt%, less than or equal to 0.8 wt%, less than or equal to 0.7 wt%, less than or equal to 0.6 wt%, less than or equal to 0.5 wt%, less than or equal to 0.4 wt%, less than or equal to 0.3 wt%, less than or equal to 0.2 wt%, less than or equal to 0.1 wt%, or less. Combinations of these ranges are also possible. For example, the surfactant may be present in the dehydrating fluid in an amount of greater than or equal to 0 wt% and less than or equal to 1 wt%, or greater than or equal to 0 wt% and less than or equal to 0.8 wt%. Other ranges are also possible.

[0088] In some embodiments, the method comprises forming a solid particle comprising the protein and / or biologic. For example, removing at least some of the carrier fluid to the dehydrating phase, as described above, may result in the formation of a solid particle. In certain embodiments, the carrier fluid and dehydrating fluid / dehydrating phase are selected in combination such that removing at least some of the carrier fluid to the dehydrating phase may increase the concentration of the protein and / or biologic and the precipitant within the remaining carrier phase (e.g., within a droplet and / or particle comprising the carrier fluid, protein and / or biologic, and precipitant). In some embodiments, increasing the concentration of the precipitant and protein and / or biologic may result in precipitant-induced precipitation of the protein and / or biologic into a solid particle (e.g., an amorphous solid dispersion comprising the protein and / or biologic and the precipitant interspersed therewith).

[0089] The solid particle may comprise the protein and / or biologic initially contained within the carrier fluid. The solid particle may also comprise the precipitant initially contained within the carrier fluid (e.g., to form a solid particle comprising the protein and / or biologic and the precipitant interspersed therewith). For example, in FIG. 3, stage 3 of method 300 shows the formation of a solid particle 320 comprising biologic and / or protein 301 and precipitant 302. The solid particle formed by this method may have any of the features, properties, and / or characteristics of a composition described above. For example, the solid particle may comprise at least some (e.g., less than or equal to 25 mg / mL) of a dehydrating fluid, which may be the dehydrating fluid of the dehydrating phase used to form the solid particle. In some embodiments, the solid particle may comprise a surfactant, which may be the surfactant contained within the dehydrating phase used to form the solid particle (e.g., the solid particle may comprise residual surfactant).

[0090] In certain embodiments, the carrier fluid may comprise one or more additional components, any of which may influence the composition and / or properties of the solution and / or suspension of the biologic and / or protein and precipitant and / or the composition and / or properties of the solid particles formed therefrom. For example, in some embodiments, the carrier fluid comprises a polymer. FIG. 4A shows an overview of a method of forming a solid particle from a carrier fluid comprising a polymer, and stage 1 of method 400a comprises providing protein and / or biologic 401, precipitant 402, and polymer 405 suspended and / or dissolved in carrier fluid 470. The polymer may be a cross-linkable polymer such as a polyalkylene oxide, a vinylsulfuone / thiol, polyethylene glycol, polyethylene glycol diacrylate, a natural polysaccharide, a modified polysaccharide, and / or alginate. The polymer may be contained within a bulk solution of the carrier fluid, biologic and / or protein, and precipitant, such that a droplet formed from said bulk solution (e.g., in the dehydrating fluid, or within a droplet-forming phase to form a pre-formed droplet) contains the polymer, as shown in stage 1 of FIG. 4A.

[0091] The polymer may be present in the carrier fluid in any of a variety of suitable amounts. For example, the polymer may be present in the carrier fluid in an amount of greater than or equal to 0.01 wt%, greater than or equal to 0.025 wt%, greater than or equal to 0.05 wt%, greater than or equal to 0.075 wt%, greater than or equal to 0.1 wt%, greater than or equal to 0.25 wt%, greater than or equal to 0.5 wt%, greater than or equal to 0.75 wt%, or greater. In some embodiments, the polymer is present in the carrier fluid in an amount of less than or equal to 1 wt%, less than or equal to 0.75 wt%, less than or equal to 0.5 wt%, less than or equal to 0.25 wt%, less than or equal to 0.1 wt%, less than or equal to 0.075 wt%, less than or equal to 0.05 wt%, less than or equal to 0.025 wt%, or less. Combinations of these ranges are also possible. For example, the composition may comprise a crosslinked polymer in an amount of greater than or equal to 0.01 wt% and less than or equal to 5 wt%, or greater than or equal to 0.05 wt% and less than or equal to 4 wt%. Other ranges are also possible. In some embodiments, the method comprises cross-linking the polymer within the carrier fluid. Cross-linking the polymer may occur during any suitable step of the methods described herein. For example, in some embodiments, the cross-linking occurs after the removing of the carrier fluid to the dehydrating phase and / or during the formation of the solid particle. In FIG. 4A, stage 2 of the method 400a comprises removing at least some of the carrier fluid 445 to dehydrating phase 480 to form the dehydrated particle 450 shown in stage 3 of the method 400a, and then cross-linking the polymer 405 to form cross-linked polymer 415 within solid particle 460 as shown in stage 4. In some embodiments, the cross-linking occurs during the removing of the carrier fluid to the dehydrating phase and / or during the formation of the solid particle. For example, stage 2 and stage 4 shown in method 400a of FIG. 4A may occur substantially simultaneously.

[0092] In some embodiments in which a pre-formed droplet comprising the carrier fluid, protein and / or biologic, precipitant, and a polymer is provided, the polymer within the pre-formed droplet may be cross-linked before the removal of at least some of the carrier fluid to the dehydrating phase. For example, in method 400b shown in FIG. 4B, a protein and / or biologic 401, precipitant 402, and polymer 405 suspended and / or dissolved in carrier fluid 470 is provided in stage 1 as droplet 490. Stage 2 of method 400b then comprises cross-linking the polymer within the droplet to form cross-linked polymer 415, and stage 3 comprises removing at least some of carrier fluid 445 to dehydrating phase 480. Stages 2 and 3 of method 400b can be performed sequentially or simultaneously (e.g., the cross-linking of the polymer and the removal of at least some of the carrier fluid to the dehydrating phase may occur sequentially or substantially simultaneously). For example, in some embodiments in which a pre-formed droplet is provided to a dehydrating fluid, the pre-formed droplet may comprise cross-linked polymer prior to the exposure to the dehydrating fluid and thus the removal of at least some of the carrier fluid thereto. In some embodiments in which the droplet is formed within the dehydrating phase, the polymer may be cross-linked while the droplet is present within the dehydrating phase (e.g., substantially simultaneously with the removal of the carrier fluid thereto). Finally, in stage 4, solid particle 460 comprising the protein and / or biologic 401, precipitant 402, and cross-linked polymer 415 is formed. In some such embodiments, when polymer contained within a droplet comprising the carrier fluid, protein and / or biologic, precipitant, and a polymer is cross-linked, a hydrogel particle may be formed. Such a hydrogel particle may then be exposed to a dehydrating phase, in which at least a portion of the carrier fluid is removed to form the solid particle comprising the protein and / or biologic. The polymer contained within the carrier fluid may be cross-linked in any of a variety of suitable ways. For example, the polymer may be cross-linked via ionic crosslinking or photoinitiated crosslinking. In some embodiments in which the polymer is cross-linked via ionic cross-linking, a droplet comprising the carrier fluid, protein and / or biologic, precipitant, and a polymer may be exposed to a secondary phase comprising an ion configured to initiate the crosslinking (e.g., a calcium ion). For example, in some embodiments, a pre-formed droplet comprising the carrier fluid, protein and / or biologic, precipitant, and a polymer may be exposed to a secondary phase comprising an ion configured to initiate cross-linking of the polymer, and then subsequently exposed to a dehydrating phase to which at least some of the carrier fluid is removed. In some embodiments, the dehydrating phase comprises the ion configured to initiate cross-linking of the polymer, such that cross-linking of the polymer within the droplet and the removal of at least some of the carrier fluid to the dehydrating phase occurs substantially simultaneously when either a pre-formed droplet comprising the carrier fluid, protein and / or biologic, precipitant, and a polymer is exposed to the dehydrating phase, or a droplet comprising the carrier fluid, protein and / or biologic, precipitant, and a polymer is formed within the dehydrating phase.

[0093] The ion configured to initiate the cross-linking of the polymer may be present in the dehydrating phase in any of a variety of suitable amounts. In some such embodiments, the ion configured to initiate the cross-linking of the polymer is present in the dehydrating phase in an amount of greater than or equal to 0 wt%, greater than or equal to 0.1 wt%, greater than or equal to 0.2 wt%, greater than or equal to 0.3 wt%, greater than or equal to 0.4 wt%, greater than or equal to 0.5 wt%, greater than or equal to 0.6 wt%, greater than or equal to 0.7 wt%, greater than or equal to 0.8 wt%, greater than or equal to 0.9 wt%, or greater. In some embodiments, the ion configured to initiate the cross-linking of the polymer is present in the dehydrating phase in an amount of less than or equal to 1 wt%, less than or equal to 0.9 wt%, less than or equal to 0.8 wt%, less than or equal to 0.7 wt%, less than or equal to 0.6 wt%, less than or equal to 0.5 wt%, less than or equal to 0.4 wt%, less than or equal to 0.3 wt%, less than or equal to 0.2 wt%, less than or equal to 0.1 wt%, or less. Combinations of these ranges are also possible. For example, the ion configured to initiate cross-linking of the polymer may be present in the dehydrating phase in an amount of greater than or equal to 0 wt% and less than or equal to 1 wt%, or greater than or equal to 0.1 wt% and less than or equal to 1 wt%. Other ranges are also possible.

[0094] In some embodiments, a method described herein comprises suspending the solid particle (e.g., formed via any of the methods described above) in an aqueous phase. The aqueous phase may have any of the features, properties, and / or characteristics of an aqueous phase of a suspension described above. For example, the aqueous phase may comprise a stabilizing agent (e.g., a stabilizing agent which may have the same composition as the precipitant of the solid particle or a different composition as the precipitant of the solid particle.

[0095] The following examples are intended to illustrate certain embodiments of the present invention, but do not exemplify the full scope of the invention.

[0096] EXAMPLE 1

[0097] Though subcutaneous (SC) delivery is the preferred administration route for immunotherapies and other biologies due to improved patient compliance and lower healthcare costs, SC administration often necessitates high-concentration antibody formulations. However, antibody solutions at high concentrations face significant instabilities and prohibitively-high viscosities. Other approaches for high-concentration formulations have been developed, including non-aqueous solutions, which can be irritating or painful, and antibody-laden hydrogel microparticles, which require centrifugation and are limited to concentrations <300 mg / mL. This Example presents the development of a formulation process in which the antibody was concentrated and encapsulated into hydrogel microparticles via solvent-based dehydration. The final dosage form was an aqueous particle suspension with concentration >350 mg / mL. In the process described in this Example, microparticles were synthesized continuously, and antibody precipitation was realized simultaneously to dehydration, which allowed for higher antibody concentrations. Antibody phase behavior and precipitation- dehydration kinetics were also analyzed. Further characterization showed antibody structure to be stable in the microparticle, and injectability of the suspension met clinical standards. An aqueous antibody formulation at high concentrations comparable to those of non-aqueous formulations is presented in this example, which is advantageous for subcutaneous administration. The process of this example is also envisioned to be generalizable as a formulation platform for SC delivery in multiple clinical applications.

[0098] 1. Introduction

[0099] Therapeutic proteins such as monoclonal antibodies (mAbs), which are widely used to treat cancers, auto-immune diseases, and other chronic and acute illnesses, are typically administered through intravenous (IV) infusion. Recently, subcutaneous (SC) injection has been emerging as the preferred administration route for mAbs due to its reduced cost and healthcare burden, enabling self-administered injections and improving quality-of-life for both patients and caregivers. However, the injection volume for SC delivery is limited (<2 mL), thus necessitating highly concentrated (>200 mg / mL) formulations to meet the dosing requirements for mAh therapies. Several strategies have been developed to enable the delivery of high-concentration biologies, which face molecular instabilities and prohibitively high solution viscosities due to intermolecular interactions. Formulating biologies as solid forms can increase the shelf-life and stability of the dosage, but can only be reconstituted in aqueous solution at low concentrations. When reconstituted into non-aqueous suspensions, both crystalline and amorphous solid mAbs have shown lower viscosities at high loadings compared to in aqueous solution. But nonaqueous formulations, typically solids suspended in an organic solvent, are limited by clinical considerations such as pain and inflammation at the injection site that is especially pronounced for SC injection, as well as regulatory hurdles to be approved for parenteral use. Thus, an aqueous formulation that maintains the advantages of stability and lower viscosity of the solid suspensions is ideal. Previous work has developed solid (crystalline or amorphous) antibodies which are loaded at high concentrations into alginate hydrogel microparticles and suspended in aqueous solution with poly(ethylene) glycol (PEG), a well-established parenteral excipient. PEG induces controlled precipitation of the protein by steric exclusion effects, and the precipitated solids can then be mixed with alginate and synthesized into hydrogel microparticles. The lubricious hydrogel microparticle reduces viscosity of the formulation and hydrogel matrix masks protein-protein interactions which are problematic for stability and injectability.

[0100] Suspending the microparticles in PEG solution ensures that the antibodies are maintained in their solid dispersion form within the hydrogel microparticle. However, the formulation concentrations achieved thus far (~ 150-300 mg / mL) have been limited by the synthesis process, which requires centrifugation to concentrate the precipitated antibodies and to produce the microparticles (FIG. 5A). Centrifugation, as a batch process, is not scalable, and it is inefficient for concentrating the aqueous suspensions which are the precursor to the hydrogel microparticles. In addition, the particles generated through centrifugal dripping are limited to >100 micrometers in diameter, while smaller particles may be desired for ease of injection or for different therapeutic applications.

[0101] Solid protein microparticles have also been formed through solvent-based dehydration. The seminal example is the Microglassification technique developed by Needham and colleagues, where protein microdroplets were dehydrated in an organic solvent to form ultra- high concentration solid microbeads (FIG. 5B). Further work showed that this technique was a viable alternative to lyophilization to form solid proteins that could be reconstituted at low concentrations in solution, or delivered at high concentrations with a non-aqueous carrier. Other groups used similar solvent-based dehydration techniques to make solid protein microparticles with improvements to particle polydispersity and process scalability, which were both issues highlighted in the prior works. However, none of these aforementioned formulations were suitable for aqueous reconstitution at high concentrations. These solid proteins are not stable in aqueous solution and would redissolve, resulting again in the issues that high concentration protein solutions face.

[0102] The aim of the work in this Example awas to develop a new formulation process for highly concentrated (>350 mg / mL) antibodies which can be encapsulated into hydrogel microparticles and produced in a continuous fashion. The final formulation consisted of antibody amorphous solid dispersion (ASD)-laden alginate microparticles suspended in an aqueous PEG solution, where the solid antibody is uniquely stable in aqueous suspension at high concentrations. The antibody is precipitated into an ASD in situ through a solvent-based dehydration process, where antibody-PEG droplets are emulsified and dehydrated in pentanol, thus becoming concentrated into high-concentration ASD-containing microparticles (FIG. 5C). The droplets are initially a stable single phase but upon dehydration the PEG and protein concentrations are driven into a region of their phase diagram where ASD formation spontaneously occurs. Alginate can be added to the dispersed phased and calcium to the continuous phase to trap the ASD inside a hydrogel matrix. The ease and scalability of microfluidic processing was leveraged to yield hydrogel particles with tight control over a broad range of accessible particle sizes. The process resulted in an aqueous hydrogel microparticle formulation with desirable flow properties at high antibody concentrations. This formulation platform is envisioned to be generalizable to multiple therapeutic modalities and dosage or encapsulation forms.

[0103] 2. Results and Discussion

[0104] 2.1 Antibody phase behavior

[0105] Given that the dehydration process in the formulation scheme described in this Example involves ASD precipitation, the phase behavior of human IgG was studied as a function of PEG concentration, at ambient temperature. In this Example, IgG was chosen as a model antibody drug as most clinically-approved antibodies are IgG types. PEG is commonly used to induce phase separation of proteins, where excluded-volume effects result in attractive depletion interactions that cause the proteins to aggregate and form a condensed phase. The exact phase that is formed can depend on several factors, including PEG and protein concentrations, pH, and ionic strength. However, in most PEG-induced precipitation studies, the different types of precipitates are often not distinguished from one another as there may only be one phase transition of interest, whether it is liquid-liquid phase separation (LLPS) for determining protein solubility, or crystallization for protein purification. But for the formulation of solid proteins, distinguishing liquid-liquid and liquid-solid phase separation is important, due to colloidal instabilities in the protein’s liquid phases. Because solid proteins are typically in the form of dry powders and precipitation is generally not desired for protein formulations, PEG-induced precipitation has not yet been well-studied in the context of solid protein formulation.

[0106] Here, the phase diagram for IgG at various PEG and protein concentrations was determined and is shown in FIG. 6A. Buffer conditions were consistent with the desired formulation. A solubility curve for IgG was constructed at various PEG concentrations (FIG. 11). Details distinguishing the LLPS and amorphous solid dispersion (ASD) precipitates are also available in the “Additional Materials” section below (FIG. 12). In this case, ASD refers to the suspension of amorphous solid antibodies dispersed in the PEG carrier. At low PEG concentrations (<3% w / v), all mixtures were stable in the liquid phase regardless of the antibody concentration (<80 mg / mL). Higher PEG concentrations induced the formation of ASDs preferentially over LLPS, with a viscous liquid condensate formed at moderate PEG concentrations and a white solid precipitate formed at high PEG concentrations, consistent with previous reports. The exact nature of the phase transition was also dependent on the protein concentration. For IgG concentrations above 40 mg / mL, 8% w / v PEG was sufficient to induce the formation of an ASD. This phase transition is reversible, as IgG will resolubilize when the PEG concentration is sufficiently low. In the proposed formulation scheme, both PEG and IgG concentration within the microdroplet would increase upon dehydration in the outer organic phase, first forming liquid-liquid separated phases and then inducing precipitation of IgG in situ into an amorphous solid microparticle.

[0107] 2.2 Single microparticle dehydration

[0108] To establish the microparticle formulation scheme, the solvent-based dehydration process discussed in this Example was studied on the single microparticle-level. Validation and improvement of the experimental setup is discussed in the “Additional Materials” section below (FIG. 13). FIG. 6B shows time-lapsed images of a single antibody microdroplet in a pentanol bath under brightfield microscopy. The microdroplet initially contained IgG and PEG, with the concentration of its components increasing as solvent-based dehydration occurred. The initial buffer concentration (5 mM) in the droplet was kept low to reduce the total salt content in the final particle. The microparticle remained spherical throughout the dehydration process due to the presence of surfactants in the surrounding organic solvent to control interfacial tension (FIG. 14A). The antibody’s phase transitions were qualitatively observed from changes in the droplet’s opacity, corresponding with the previously discussed phase behavior. Concentrations of PEG and IgG within the microparticle were also estimated by the measured change in droplet size. At t=0 minutes, IgG (60 mg / mL) was soluble at a low PEG concentration (2% w / v), as observed by the clear droplet. At t=l minute, liquid-liquid phase separation was observed from the cloudy droplet due to the second liquid phase as water was extracted. By t=3 minutes, increasing PEG concentration (—4% w / v) within the droplet induced precipitation of the IgG (-120 mg / mL) into the amorphous solid form, resulting in an opaque particle. This solid-liquid transition was primarily driven by the increasing concentration of PEG which quickly induced precipitation of the antibody. By contrast, without PEG in the initial droplet, the antibody remained in solution until reaching supersaturation and nucleating into an amorphous solid, a process that occurs on a much longer timescale than PEG-induced precipitation due to the generally high solubility of IgG (FIG. 14B). With PEG present in the initial droplet, the antibody was incorporated into an amorphous solid dispersion, which remained solid at sufficient PEG concentrations. To investigate the kinetics of the dehydration process for various initial conditions, the relative change in droplet radius was recorded over time for varying initial IgG and PEG concentration, shown in FIG. 6C and FIG. 6D, respectively. When normalized by surface area, the kinetics of the dehydration process were not affected by changes in solute concentration. In the initial time period, the kinetic data fit well to the Epstein-Plesset (EP) model before the particle plateaued to its final equilibrium size. As expected, the normalized equilibrium size of the particle was dependent on the initial concentrations of each solute, but interestingly did not show a directly proportional relationship. In fact, droplets with 40 mg / mL and 60 mg / mL inital IgG concentration (CigG.o) resulted in particles of similar size despite the difference in solids content, suggesting that packing of the amorphous antibody was more efficient in the case of cigG,o=60 mg / mL. This effect is highlighted when the final concentration of IgG (CigG,f) in the particle is estimated using the change in droplet size. As shown in FIG. 6E, CigG,f is much higher for CigG,o=60 mg / mL than the other conditions. Similarly, droplets initially with 2% and 4% w / v PEG displayed more efficient ASD packing compared to the CPEG,O=1% W / V condition, though it is noted that IgG solutions with 4% w / v PEG are not stable since the higher PEG concentration led to LLPS. The differences in ASD packing density based on the initial droplet composition could be due to increased attractive interactions in the cases of higher PEG or IgG concentration. These results suggest there are preferable conditions to achieve high ASD packing density and thus concentration, which could be a strategy for improving ultra-high concentration solid antibody formulations. Without PEG in the initial droplet (CPEG,o=0% w / v), the change in size and thus concentration was the largest due to the absence of a polymer matrix. It was estimated that estimate CigG,f within these formed particles was ~830 mg / mL, which is in the range of what Needham and colleagues had reported for various proteins. With the addition of PEG, the final particle concentrations were estimated to be between ~300-600 mg / mL (FIGS. 6E-6F). It is expected these concentrations would be lower when PEG is initially present in the droplet, due to the higher total solids content and the retention of water in the polymer matrix of the ASD. As the results described in this Example demonstrate, ASD formation is sensitive to both PEG and IgG concentrations within this range. As the final particle loading is dependent on the initial concentrations of each constituent, it is important to consider the formulation of the droplet solution. Regardless, the estimated CigG,f of these particles were still higher than what could be achieved with the previous process. Solvent-based dehydration overcame the previous limitations and enabled ultra-high concentration ASD-laden particles.

[0109] While highly concentrated solid ASD microparticles were formed via this dehydration process, the resulting particles were brittle and not mechanically robust. In addition, it is known from previous works that encapsulating antibody ASDs into hydrogel particles facilitates the stability and injectability of the formulation, due to the hydrogel masking protein-protein interactions and lending its lubricious rheological properties to the formulation. Therefore, encapsulation of the antibody ASD into hydrogel microparticles was sought, concurrent to the dehydration process. To make cross-linked hydrogel microparticles, alginate polymer (0.2% w / v) was dissolved in the antibody solution, and calcium chloride was dissolved in the outer pentanol phase. Alginate cross-links ionically via association with calcium cations, leading to the formation of an alginate scaffold which encapsulates the solid antibodies as dehydration and protein precipitation occur (FIG. 7A). As proposed, these alginate microparticles can be resuspended into an aqueous solution with PEG to stabilize the solid antibody within the hydrogel. This process was first shown for individual alginate microparticles loaded with IgG (FIG. 7B). First, the droplets are simultaneously precipitated and cross-linked in a pentanol bath, forming a opaque, concentrated solid microparticle. Then, the particles are transferred to an aqueous solution with 15% w / v PEG (buffered with 5 mM HEPES, pH 7.4). IgG remains encapsulated in the microparticle in its solid form as PEG will prevent dissolution of the antibody. It was also shown that this stabilization mechanism is due to PEG and does not depend upon the particle’s alginate content (FIG. 15). Though including PEG in the initial droplet solution limited the particle loading, it enabled the formation of a stable ASD which allowed for aqueous reconstitution of the particles. Finally, these particles are transferred into a simulated body fluid (SBF), where the drop in local PEG concentration will cause IgG to dissolve and diffuse from the particle, leaving behind the alginate scaffold. Without a sufficient PEG concentration, IgG will resolubilize, thus resulting in the blank hydrogel particles shown in FIG. 7B. The antibody’s phase behavior here demonstrates that the antibody can be viably delivered in its solid form in an aqueous vehicle and that its precipitation is reversible.

[0110] The kinetics of the dehydration process for the alginate hydrogel microparticles was also investigated. For droplets containing alginate, there was a slight increase in the normalized equilibrium particle size compared to those droplets with only IgG and PEG, owing to the additional polymer content in the particle (FIG. 7C). However, the concentration of calcium cross-linker in the pentanol bath did not affect either the kinetics or the equilibrium behavior of the droplets, with a final estimated concentration factor (Ro3 / Rf3) of ~6x for all conditions (FIG. 16). The cross-linking of alginate forms a solid hydrogel network which does not contribute to volume exclusion of the protein, so the cross-linked alginate would ultimately not affect the antibody’s phase behavior. These results showed that ASD-laden hydrogel particles could reliably be produced and resuspended in various aqueous media with predictable behavior, which is important for its potential clinical applications.

[0111] 2.3 Continuous microparticle encapsulation

[0112] For application of the formulation, production of the ASD-laden hydrogel particles in a scalable manner was sought. To demonstrate scalability of this solvent-based dehydration process for solid mAb formulation, continuous encapsulation of antibody into alginate microparticles via solvent-based dehydration was accomplished via a microfluidic cross-junction (FIG. 8A). IgG solution containing PEG and alginate was dispersed in a continuous pentanol phase, with the dispersed droplets precipitating and crosslinking into hydrogel microparticles. Due to the immiscibility of the two fluid phases, antibody droplets were easily formed via pinch- off in the continuously flowing pentanol phase. Surfactant (Tween 80) was used in the continuous phase to control interfacial tension. The flow rate of the dispersed phase was held constant at Qd =4 microliters / min while the flow rate of the continuous phase (Qc) was varied to influence the hydrogel particle size (FIG. 8B). As the flow rate ratio Qc / Qd increased, smaller particles were produced due to the greater shear force of the continuous phase at the crossjunction. The size of the crossjunction thru-hole (150 micrometers) also controlled the range of particle sizes that were achieved. FIG. 8C shows representative images of antibody-laden hydrogel microparticles produced at various flow rate ratios, with generally consistent particle size and sphericity. For Qc / Qd >40, particles <100 micrometers in diameter could be generated. This size range was previously inaccessible in prior work based on limitations of centrifugal synthesis. The ability to generate particles in this size range represents a significant advance over the previous process, especially for subcutaneous injection where thinner needles are desired. However, the greater shear forces at higher flow rate ratios also resulted in the fragmentation and formation of satellite particles. As such, a moderate flow rate ratio (30x) was employed to produce particles for analysis, owing to the more uniform particle morphology and size distribution at this ratio. Previous encapsulation processes for ASD-laden hydrogel particles relied on centrifugation, which was neither continuous nor scalable. This encapsulation process was able to continuously generate microparticles with a wide range of characteristic diameters suitable for injection and moderately tight control over particle size. The droplet-based microfluidic setup used for this process can be easily scaled up for high-throughput applications. More advanced microfluidic designs could be utilized to increase output and meet other particle size specifications.

[0113] The continuous microfluidic encapsulation process was used to produce antibody ASD- laden microparticles for further characterization. Particles of 105+16 micrometers diameter were used for characterization of particle loading and the further analysis described later in Section 2.4. To determine the final formulation concentration, particle loading was measured by determining the sample volume of antibody-laden particles and eluting antibody from the particles to measure total IgG mass in the particles. Microparticle loading is dependent on the initial IgG solution concentration (FIG. 15A). For these particles with initial conditions CigG,o = 60 mg / mL and CPEG.O = 2% w / v, the particle loading was calculated to be 486+35 mg / mL (n=3), an ~8x increase in concentration which is a similar concentration factor to what was predicted in the single microparticle experiments. These initial concentrations of IgG and PEG were selected for the most efficient ASD packing with an initially stable IgG solution, based on the phase behavior and single-particle experiments. The final particle loading was much higher than what could be achieved with previous processes which relied on centrifugation to concentrate and encapsulate the ASD. The current process enabled continuous particle production and resulted in higher particle loadings, which represent significant improvements towards a platform for SC formulations. Initial droplet compositions could potentially be tailored to reach higher microparticle loadings, which would involve further studies on ideal conditions for ASD formation and packing. However, the current particle loading approaches the theoretical limit suggested by Garidel et al. (500 mg / mL), indicating that packing efficiency within the particle is already quite high. Encapsulation efficiency (E.E.) of the process was defined as the mass of encapsulated antibody over the total mass of antibody in solution. To measure the E.E., the amount of IgG in pentanol bath after particle generation compared to the amount of IgG in the initial solution. The E.E. for the continuously generated microparticles was high (98.8+0.2%), due to minimal leaching of the antibody from the droplets. Limited leaching of antibody was expected to occur during the dehydration process as the antibody is not soluble in pentanol. This E.E. is comparable to that of prior processes for antibody-laden alginate microparticles, and is much higher than those of typical protein encapsulation processes.

[0114] An important limitation of previous formulation approaches for solid antibody formulations was the inability to resuspend these solids in aqueous phases, either requiring reconstitution in solution at low concentrations or delivery in non-aqueous vehicles which pose regulatory and manufacturing challenges. After continuously generating antibody-laden hydrogel microparticles, it was demonstrated that these particles are easily resuspendable and stable in aqueous solution, facilitating delivery in an aqueous vehicle. Due to the formation of the amorphous solid dispersion via PEG-induced precipitation, the antibody was retained in its solid form even in aqueous solution as long as PEG was present in sufficient concentrations. This effect was qualitatively shown earlier for antibody-laden alginate microparticles FIG. 7B). The apparent solubility of IgG from the particles in aqueous solutions with varying PEG concentration is shown in FIG. 8D. Clearly, without PEG in solution (0% w / v), the antibody has a tendency to diffuse from the particle as previously discussed, resulting in a relatively high apparent IgG solubility. However, antibody solubility steadily decreases as the concentration of PEG in solution increases, which is expected as PEG is known to systematically decrease protein solubility. In fact, apparent IgG solubility in 10% and 15% w / v PEG solution is very low, 0.03 mg / mL and 0.01 mg / mL respectively. Therefore, a 10% w / v PEG solution was sufficient to stabilize IgG in its solid form and prevent dissolution of antibody from the particles. This result is consistent with the phase diagram presented earlier (FIG. 6A), where 10% w / v PEG induced and stabilized ASD formation at all protein concentrations. In this manner, the antibody-laden particles can be delivered as an aqueous suspension rather than using nonaqueous carriers as other high-concentration formulations. The ability to deliver high-concentration antibodies in aqueous suspension offers an advance over the state-of-the-art. Non-aqueous suspensions lead to increased injection site pain, toxicity, and pose manufacturing complications and a regulatory burden that this approach overcomes.

[0115] 2.4 Applicability as a formulation platform Further characterization of the antibody-laden microparticles was carried out to support the applicability of the formulation process as a potential platform for subcutaneous delivery of antibodies. First, injection force tests were performed on the final aqueous particle suspension to assess injectability of the formulation. Compared to other material properties such as viscosity and storage and loss modulus, injection force is the most relevant measurement for clinical applications and yields interpretable quantitative results. Injectability is typically a challenge for SC delivery due to the viscosity of high concentration antibody solutions. Lower injection forces are desired for viability and ease of administration.

[0116] The tests were performed using a universal mechanical tester to apply a downward compressive force on the syringe plunger over a specified displacement. A particle suspension with a formulation concentration (Cform) of 360+9 mg / mL IgG was used, depicted in FIG. 9A. The Cform is based on the previously calculated particle loading and a particle volume fraction (Vparticies / Vparticies+PEGsoiution) of 0.75 in 10% w / v PEG solution, confirmed by dissolving the formulation in phosphate-buffered saline after performing the injection tests and measuring the supernatant concentration via UV-vis. This Cform represents a new upper limit in ultra-high concentration aqueous antibody formulations, where previous formulations were limited to <200-300 mg / mL, due to the dehydration process which overcame the concentration limitations of the previous processes. The increase in Cform is significant as higher concentrations enable lower volume doses or less frequent injections, both of which are favorable for accessibility and patient compliance. The formulation was injected through a 25-gauge needle, which is standard for subcutaneous injection. A flow rate of 25 microliters / s was selected for the tests as a moderate, clinic ally -relevant injection speed. FIG. 9B shows the average injection force profile over the syringe plunger displacement. In the beginning of the test, the microparticle formulation experiences a ‘start-up’ regime where the injection force increases until it reaches a plateau, which is typical for injection force tests. In the plateau regime, there are still variations in the injection force profile as the plunger moves, both within and across individual tests. This suggests that there are local differences in the distribution of the particles and the injection force may experience some dependence on how the formulation is loaded. The force profile in this plateau regime can be averaged to yield the glide force, which ranged between 10 and 20 N for the tests. These glide forces were comparable to those reported for non-aqueous mAb suspensions at similar antibody concentrations. Regardless of the variations, the injection force profiles for all tests were well-below the suggested limit of 40 N as the maximum acceptable force for subcutaneous injection. Also, the same injection tests for an IgG solution (150+4 mg / mL) as a control resulted in comparable glide forces as the microparticle formulation, even though the microparticle formulation was >2 times more concentrated than the solution. A stable IgG solution at a higher concentration was not possible for us to make. The relatively low injection forces of the microparticle formulation can be attributed to the encapsulation of the antibodies into hydrogels, which lend advantageous rheological properties, such as particle deformability and shear-thinning behavior and help to improve injectability. Ease of injection is key for SC delivery to enable self-administration and home-based care which are touted as major benefits of SC delivery. These results show that the particle formulations can be injected at ultra- high concentrations with well-accepted injection forces. Thus, the formulation described in this Example is viable for self-administration, which can improve accessibility, patient compliance, and quality-of-life.

[0117] To show the structural stability of the antibody after dehydration and encapsulation, characterization of the antibody secondary structure was performed via both far-UV circular dichroism (CD) and FTIRATR spectroscopy. Following microparticle generation, the particles were removed from the organic solvent and the antibody was eluted into solution for analysis. The far-UV CD data (FIG. 9C) showed comparable mean residual ellipicity between native IgG reference and the reconstituted IgG from the microparticles, indicating that the structure and conformation of the antibody was not irreversibly affected by the dehydration-encapsulation process. FTIR spectroscopy was used to quantify the secondary structural composition, via deconvolution and analysis of the amide I band region (1700-1600 cm-1). The estimated compositions of the native and reconstituted IgG are shown in FIG. 9D. Representative FTIR spectra are available in the Additional Materials section (FIG. 17). Both IgG conditions were consisted of majority P-sheet structure (~60%), which is consistent with IgG antibody literature. The percent composition of P-turns (~30%) and a-helices or random coils (~ 10%) were also in the range of previous reports, and there were no significant differences between the native reference and reconstituted antibody composition. While dehydration is known to typically perturb antibody structure due to the removal of water and rearrangement of intermolecular interactions, these effects were shown to be reversible for the microparticle formulation after elution of the antibody in aqueous media. PEG-induced precipitation of the antibody during the dehydration process resulted in a stable ASD, where PEG preserved the solid antibody form, given that the antibody’s native structure was recovered upon dissolution. Size exclusion chromatography was also performed to confirm that irreversible aggregation had not occurred during the dehydration process, with the antibody recovered from the particle as monomers (FIG. 19). Finally, it was demonstrated that the antibody could be easily released from the particles upon injection in a physiological media. Release of the antibody from the alginate microparticle was studied in vitro at physiological conditions using simulated body fluid (SBF) as the release media. The release profile for IgG at 37 °C is shown in FIG. 9E. The release of the antibody from a microparticle submerged in SBF is also visualized by observing a decrease in the opacity of the particle over time. Upon injection into SBF, the local PEG concentration in the microparticle decreases, which triggers dissolution of the antibody and diffusion from the particle, as described earlier. This process occurs rapidly, with most of the antibody (~80%) released within the first 10 minutes of the test, which is comparable to other reports for high-concentration solid antibody formulations. Fast release of the antibody is common for hydrogel-based delivery systems, due to the high permeability of water in the hydrogel. Altogether, characterization of the ASD-laden microparticles indicates that the formulation has acceptable injectability, stability, and release properties, advancing this solvent-based dehydration process as a viable platform to formulate high-concentration solid antibodies.

[0118] 3. Conclusion

[0119] Subcutaneous administration of therapeutic antibodies requires high-concentration formulations for reduced injection frequency or volume and improved patient compliance and quality-of-life. In this work, an ultra-high concentration (360 mg / mL) aqueous antibody formulation with acceptable injectability and stability properties is presented. Solvent-based dehydration was applied to concentrate antibody droplets and trigger an in situ PEG-induced phase transition from liquid to solid antibody, using IgG as a model drug. Amorphous antibodyladen hydrogel microparticles were continuously synthesized through a facile microfluidic process which can be modulated to achieve different output parameters such as particle size. The microparticles were suspended in PEG solution where the antibody was retained in its solid form, uniquely combining the stability of a solid antibody formulation with the benefits of an aqueous dosage form. Due to the advantages of hydrogels for therapeutic encapsulation and delivery, the final formulation was able to be injected at a high concentration, while showing rapid release in a simulated body fluid. Overall, the results of this work demonstrate the potential of the ASD microparticle formulation as a platform to formulate high-concentration antibodies for SC delivery. In addition, it is expected for this process to be generalizable, both in terms of the therapeutic modalities and the dosage form. While the process was validated here for an amorphous antibody, other solid states (i.e. crystalline, coacervate) would be also suitable for formulation. It is viable for the platform to be expanded to other therapeutic molecules (i.e. mAbs, peptides, nucleic acids) as long as the molecule can be stabilized in its solid form, whether via PEG or another precipitant. The encapuslation process could also be modified with different polymers and cross-linkers to design hydrogel particles with desired rheological properties or release profiles.

[0120] 4. Experimental Section

[0121] Materials:

[0122] All chemicals used were of analytical grade. Lyophilized total human IgG was purchased from Equitech-Bio, Inc. Poly(ethylene) glycol (PEG, 3350 kDa) was purchased from Rigaku Reagents. Sodium alginate (5 - 40 cP) was purchased from Sigma. All other chemicals were purchased from Sigma and used without further purification.

[0123] Phase transitions in IgG solutions: To study PEG-induced phase separation in IgG, concentrated PEG solution (50% w / v) was added dropwise to a stock IgG solution, prepared from the lyophilized product, while stirring. The mixtures were buffered at pH 7.4 with 5 mM HEPES (N-2-hydroxyethylpiperazineN-2-ethane sulfonic acid) and was carried out in batches at a total volume of 0.3 mL. All solutions were filtered with a 0.2 micrometers filter before mixing. The resulting mixture was kept at room temperature for 1 h while rotating at 20 rpm on a tube mixer. Afterwards, the mixture was centrifuged at 1700 RCF for 30 minutes to recover any precipitates. To determine the solubility of IgG at various PEG concentrations, the protein concentration in the supernatant after centrifugation was measured in a UV-vis Nanodrop spectrophotometer using the 280 nm absorbance method. To distinguish between liquid-liquid and liquid-solid phase transitions, the appearance of the precipitates was evaluated.

[0124] Single microparticle dehydration experimental setup: Single droplets of antibody solution (0-60 mg / mL) containing PEG (0-4 were expelled from a syringe with a 30G stainless steel needle (I.D.= 159 micrometers, O.D.= 312 micrometers) into a 20 mL 1-pentanol (0.4% w / v Tween 80) reservoir. The pentanol reservoir was held in a glass-bottom Petri dish spin-coated with polystyrene to hydrophobize the bottom surface and minimize contact of the microdroplet with the dish. The Petri dish was mounted upon an inverted optical microscope equipped with a digital camera to record the dehydration of single microparticles. Frames from the captured video were analyzed using ImageJ to measure the droplet diameter during dehydration and the size of the dehydrated microparticle. All experiments were performed at ambient temperature with an initial microdroplet radius between 150 to 200 micrometers, with n=3 for each condition. Continuous antibody encapsulation: IgG ASD-laden alginate microparticles were produced continuously using a microfluidic cross-junction (IDEX PEEK P-891, thru hole=0.15mm, O.D.=l / 16in) with softwalled tubing (PTFE, I.D.=0.3mm, O.D.=l / 16in). The cross-junction consisted of a cross-shaped channel intersection with four total inlet and outlet ports (FIG. 8A). The dispersed phase containing aqueous antibody solution (40-60 mg / mL) with PEG (1-4 w / v%), sodium alginate (0.2 w / v%), and 5 mM HEPES (pH 7.4) is introduced through one inlet, while the continuous phase consisting of 1-pentanol with calcium chloride (0.01 wt%) and Tween 80 (0-0.4 wt%) is delivered through the two inlets perpendicular to the dispersed phase. The solutions are loaded into syringes and connected to separate syringe pumps (Harvard Apparatus PHD 2000). The flow rate of the dispersed input was controlled at 4 microliters / min while the flow rate of each continuous input was varied between 40 to 320 microliters / min. To minimize pressure variations and ensure reproducible results, all tubing and the microcross are secured flat against a level surface. The microparticle samples were collected at steady state in a pentanol bath at the outlet port. Images of the microparticles were taken after complete dehydration under brightfield microscopy. The size distribution of the microparticles was analyzed for each condition using the StarDist object detection method to obtain final particle diameter (n>30).

[0125] Antibody particle stability in PEG solution: For evaluating the apparent solubility of the amorphous solid antibody in suspension with PEG to ensure the stability of the solid phase in the aqueous phase, IgG ASD-laden alginate microparticles were generated via the microfluidic process described. The samples were transferred to a microcentrifuge tube and excess solvent was removed to adjust the total IgG content in each tube to 1 mg. 1 mL of storage buffer (5 mM HEPES, pH 7.4) with different w / v% concentrations of PEG was added each tube and the samples were left to equilibrate with the storage buffer at room temperature (~22°C). After 24 h, the protein concentration in the supernatant was measured using the 280 nm absorbance method.

[0126] Injection force measurements: The injectability of the microparticle formulation was quantified using injection force measurements according to a previously described protocol. A Zwick- Roell universal testing machine (model Z010) equipped with a 500 N load cell and custom 3D-printed compression test flat-plate attachment was used. All tests were performed at ambient conditions with multiple sample replicates (n=3). The particle suspension formulation was loaded into a BD 1-mL syringe with a 25-gauge (ID= 260 micrometers, OD= 515 micrometers) Luer-lock needle. The syringe was clamped in place during the test. For each experiment, the stroke distance (displacement) was controlled at 25 mm, corresponding to a ~0.5 mL injection volume. The stroke speed was set at —1.4 mm / s, which corresponds to a injection rate of 25 microliters / s.

[0127] Circular dichroism (CD): Far-UV circular dichroism (CD) was used for IgG secondary structure determination, using a JASCO J-1500 spectropolarimeter. IgG samples were prepared at 0.5-1 mg / mL in aqueous solution (5 mM HEPES, pH 7.4), and spectra were obtained in a 1 mm quartz cuvette (Hellma Analytics) at ambient conditions. The wavelength was scanned from 250-190 nm with a 0.5 nm data pitch, 1 nm bandwith, 4 s digital integration time, and 50 nm / min scanning speed. A nitrogen gas purge was run for at least 5 minutes before sample measurement. The spectra recorded were the average of five individual background-subtracted scans, and the data was reported as mean residual ellipticity following the protocol in Kelly et al.

[0128] Fourier transform infrared spectroscopy (FT1R): Solvated and solid state samples of IgG were analyzed using FTIR spectroscopy (Thermo Fisher Nicolet is50) with a built-in diamond ATR crystal. Either a 20 microliters drop of IgG sample, 40 mg / mL concentration in simulated body fluid (SBF) for solvated samples or a powder for solid state samples was placed on the ATR crystal. 256 scans were collected at a resolution of 4 cm-1, with the background spectrum automatically subtracted. The spectra were analyzed using Fityk software. The amide I band region (1700-1600 cm-1) was isolated, baseline-corrected, and area-normalized. The second derivative spectra was obtained using 9-point Savitzky-Golay smoothing algorithm and peak- fitted with a Gaussian curve function and the Levenberg-Marquardt algorithm. Peaks were assigned to secondary structures following the literature conventions. The relative areas of the fitted peaks were used for the quantification of IgG secondary structure (n=5 for each sample).

[0129] In vitro release assays: To evaluate release of the antibody from the hydrogel microparticle, approximately 20 microliters of the microparticle suspension was injected into a 2 mL centrifuge tube filled with 1 mL of pre-warmed (37°C) simulated body fluid (SBF), which was prepared based on the literature to mimic the ionic composition of the SC environment, comprised of 7.996 g / L sodium chloride, 0.350 g / L sodium bicarbonate, 0.224 g / L potassium chloride, 0.228 g / L potassium phosphate dibasic trihydrate, 0.305 g / L magnesium chloride hexahydrate, 0.278 g / L calcium chloride, 0.071 g / L sodium sulfate, 6.057 g / L tris (hydroxymethyl) aminomethane, and 40 mL / L of 1 M hydrochloric acid. At specified time intervals, 200 microliters of the supernatant was removed and taken for measurement of protein concentration via the 280 nm UV-vis absorbance method, and the sampled volume was replaced with fresh SBF. Measurements were taken in triplicate using multiple sample replicates (n=3).

[0130] Additional Materials IgG phase behavior and solubility

[0131] To determine the solubility of IgG, the concentration of IgG in the supernatant was measured using an UV-vis spectrophotometer for different precipitation conditions. The concentration of the supernatant was then taken as the apparent solubility of IgG, S. FIG. 11 shows log(S) at varying PEG and IgG concentration, coded either by initial IgG concentration or the condensed phase. Regardless of the nature of the condensed phase or the initial protein concentration, IgG solubility exhibited characteristic linear dependence of log(S) on the PEG concentration. Extrapolating from the linear fit, the apparent solubility of IgG at these conditions (5 mM HEPES, pH 7.4) for zero PEG concentration is ~215 mg / mL (log(S) = 5.4). The log(S) value at zero PEG is within the range of other reported apparent solubilities for IgG-type antibodies, with variations based on pH and ionic strength. It is clear though that for CIgG > 20 mg / mL and CPEG > 8% w / v, the condensed phase that formed was an amorphous solid rather than liquid phase.

[0132] In addition, FIGs. 12A-12B show qualitatively the differences observed between LLPS and ASD cases. LLPS was observed as dispersed droplets in a second liquid phase under microscopy and as a clear or translucent precipitate, whereas ASD was observed as fractal-like solid protein aggregates under microscopy and as a opaque white precipitate.

[0133] Single droplet experimental setup validation

[0134] The Epstein-Plesset equation was first developed in 1950 for the dissolution rate of a gas bubble in liquidgas solution: dR __ __ f j. 1 ] dt ( ’ (i) where a = Dcs / p(l - f), f = ci / cs, and ci, cs being the initial and saturated concentration in the bulk phase. The initial saturation ratio of pentanol, f, was assumed to be 0 for these experiments. The dissolution rate of water droplets in an organic phase can generally be successfully modeled using the Epstein-Plesset (E-P) model, including with solute- and protein-containing droplets. The value of parameters for the E-P model in the experimental system is tabulated below in Table 1.

[0135] Table 1. Relevant parameters for Epstein-Plesset model.

[0136] To validate the experimental setup for this work, droplets of pure water (Ro = 150 - 200 micrometers) were generated using the setup described in the main text, and the rate of dissolution was fitted to the E-P model. The diffusion coefficient, D, was extrapolated from fitting the kinetic data. A correction factor of 0.72 was applied based on previous studies to account for the droplet’s contact with the bottom surface of the Petri dish. Using this experimental setup, values of the diffusion coefficient for water in pentanol (0.50 ± 0.04 * 10“5cm2 / s) were found that were comparable to those in the literature, thus validating the current setup. The data and fitted models are shown below for the validation experiments of water droplets in 1-pentanol (FIG. 13). As shown, the data displayed good fit to the model, with R2 values ranging from 0.933 - 0.978. In addition, when scaled by tf: which is the theoretical time for a water droplet of initial size Ro to completely dissolve, the data collapse on one master curve independent of initial droplet size. Therefore, the fitted diffusion coefficient in the E-P model was able to be applied to compare to the single-droplet experiments in the main text for antibody-laden droplets.

[0137] Single droplet experiments without surfactant

[0138] Without surfactant (Tween 80) in the pentanol phase, the antibody droplets tended to form non-spherical and irregular morphologies. A set of time-lapsed images of an antibody solution droplet in pentanol without surfactant is shown in FIG. 14A. Although dehydration of the droplet and increasing PEG and IgG concentration induces precipitation of the antibody, with or without surfactant, the particle that is formed without surfactant present is misshapen with rough surface features that could affect the flowability of the particles. Therefore, surfactant was used in the pentanol phase to consistently generate smooth, spherical microparticles by controlling interfacial tension.

[0139] Single droplet experiments without PEG

[0140] As described in the main text, single droplet experiments using the validated experimental setup were performed for antibody solution droplets (CIgG,o=60 mg / mL) without PEG (FIG. 14B). Because of the absence of PEG or other polymer, the sole driving force for the antibody liquid- solid transition was the extraction of water from the droplet. During the dehydration process, the antibody is observed to remain in solution as a clear droplet until t=~7 min, which is approximately the time it takes for the droplet to be concentrated above the solubility limit for IgG. At that time, the droplet has experienced a 300-400% decrease in its volume due to dehydration, causing IgG to reach supersaturation and precipitate out of solution into its solid form. By contrast, with PEG (2% w / v) in the initial solution, IgG begins to liquidliquid phase separate with only a ~30% decrease in droplet volume and fully precipitates for a ~100% decrease in volume. Clearly, PEG when present in the initial droplet is the major driving force for protein precipitation, and it is also key for stabilizing the protein as an amorphous solid.

[0141] Stability of antibody microparticles without alginate

[0142] As discussed in the main text, solid antibody remains encapsulated in a hydrogel microparticle in an aqueous phase due to the presence of PEG in the solution, which prevents redissolution of the antibody. This effect is consistent even without the alginate hydrogel. Particles were produced via dehydration of antibody-laden droplets in pentanol, without alginate in the initial solution, and then resuspended in a 15% w / v PEG solution. From FIG. 15, it was observed that the microparticles remain opaque in the aqueous PEG solution, showing that PEG is able to stabilize the solid antibody as expected, and this mechanism is not dependent on an hydrogel network.

[0143] Estimated final particle concentration in single droplet experiments

[0144] For the single droplet experiments, the final IgG concentration (CIgG,f) in the particle following the dehydration process could be estimated based on the observed change in droplet size and the initial IgG concentration in the droplet. The ratio of the initial and final droplet sizes can be taken as the concentration factor:

[0145] CigG,f= (Ro / Rf) *CigG,o (3)

[0146] The calculated CigG,f are shown in FIG. 16 for various calcium concentrations in pentanol, tested in the single droplet experiments with 60 mg / mL IgG, 2% w / v PEG, and 0.2% w / v alginate initially in the droplets. Variations in CigG,f between different conditions are discussed in the main text.

[0147] FTIR spectra Representative second derivative FTIR spectra for the amide I band region are shown below for the native reference and the reconstituted IgG (FIG. 17). The second derivative spectra were used for quantification of the antibody secondary structure, which is a standard technique for FTIR spectroscopic analysis to enhance the resolution of FTIR measurements.

[0148] Size exclusion chromatography (SEC)

[0149] Analytical SEC was used to determine the quantity of IgG monomer and aggregates from eluted from ASD-laden alginate microparticles. For this purpose, an Agilent 1200 HPLC instrument was used, with a TSKgel G3000SWXL (Tosoh Bioscience) analytical SEC column. SEC experiments were carried out at a flow rate of 0.4 mL / min in phosphate buffered saline (PBS) at pH 7.4. For the native control experiment, lyophilized IgG powder as received was dissolved into PBS and analyzed. The SEC results from integrating the signal are tabulated below in Table 2. Experiments were performed in triplicate (n=3) for each condition, with standard deviations reported.

[0150] Table 2. Stability of IgG evaluated using size exclusion chromatography.

[0151] As seen in Table 2, the quality of the IgG released from the alginate microparticles is not significantly different from the native control (>90% monomer), indicating that IgG remains stable and does not irreversibly aggregate when formulated into the hydrogel via the solventbased dehydration process. Characteristic UV traces for each condition are available in FIG. 18.

[0152] The following examples demonstrate the production of high-concentration protein microparticles via solvent-based dehydration in the presence of polyethylene glycol (PEG), resulting in stable, flowable microparticles that retain their integrity when resuspended in an aqueous solution. These examples illustrate both the single and co-formulation of proteins into microparticles using scalable, continuous processes that do not require centrifugation

[0153] Example 2. High-Concentration Precipitation of Proteins with PEG

[0154] As depicted in FIG. 19, the high-concentration precipitation of immunoglobulin G (IgG) was achieved by exposing an aqueous IgG droplet to an immiscible solvent, specifically 1- pentanol. To investigate the impact of PEG on particle formation, two different IgG solutions were prepared: 40 mg / mL IgG with 4% PEG; and 40 mg / mL IgG without PEG (0% PEG).

[0155] Droplets of each solution were placed on a glass slide flooded with 1 -pentanol and monitored over time. During the dehydration of the droplet, the protein was precipitated in situ due to increasing concentration of both protein and PEG in aqueous droplet. The droplets containing PEG transitioned to an opaque appearance, signifying the formation of an amorphous solid microparticle (FIG. 20A). In contrast, droplets without PEG did not undergo opacity changes (FIG. 20B).

[0156] Example 3. High-Concentration Precipitation with PEG and a Polymer Binder

[0157] The role of PEG in combination with a polymer binder, alginate, was also explored to produce stable microparticles. An initial solution containing IgG (10 mg / mL), PEG (2%), and alginate (0.2%) was prepared. Upon exposure to 1 -pentanol, the solution underwent volume reduction, concentrating the IgG to approximately 950 mg / mL within the microparticle. The final composition in droplet comprised 40 mg / mL IgG, 1% w / v PEG, and 0.2% w / v alginate (FIG. 21 A). The final protein concentration was -950 mg / mL (24X volume reduction) (FIG. 21B).

[0158] Example 4. Co-Formulation of Multiple Proteins

[0159] A co-formulation of two proteins into the same microparticle was achieved using PEG- based precipitation. The following initial protein solutions were used: 40 mg / mL bovine serum albumin (BSA), 4% PEG (FIG. 22A); and 20 mg / mL IgG, 20 mg / mL BSA, 4% PEG (FIG. 22B).

[0160] Droplets of each solution were dehydrated in 1 -pentanol, forming co-formulated microparticles. The resulting protein particles maintained a defined stoichiometry reflective of the initial droplet composition.

[0161] Example 5. Resuspension of Protein Particles in Aqueous PEG Solution

[0162] The stability of the dehydrated protein particles when resuspended in an aqueous solution was evaluated. FIGs. 23A and 23B demonstrate this stability. In FIG. 23 A, IgG particles (40 mg / mL IgG, 4% PEG) were initially emulsified and dehydrated in 1 -pentanol. Following dehydration, the particles were washed with ethanol. In FIG. 23B, the particles were resuspended in a 25% PEG aqueous solution, where they formed a uniform suspension without dissolving. This resuspension ability confirms that the protein microparticles remain as flowable solids.

[0163] While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present invention is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and / or methods, if such features, systems, articles, materials, and / or methods are not mutually inconsistent, is included within the scope of the present invention.

[0164] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0165] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and / or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0166] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0167] As used herein, “wt%” is an abbreviation of weight percentage. As used herein, “at%” is an abbreviation of atomic percentage.

[0168] Some embodiments may be embodied as a method, of which various examples have been described. The acts performed as part of the methods may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include different (e.g., more or less) acts than those that are described, and / or that may involve performing some acts simultaneously, even though the acts are shown as being performed sequentially in the embodiments specifically described above. Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

[0169] When a portion (e.g., a layer, a structure, a region) is “on”, “adjacent”, “above”, “over”, “overlying”, or “supported by” another portion, it can be directly on the portion, or an intervening portion (e.g., layer, structure, region) may also be present. Similarly, when a portion is “below” or “underneath” another portion, it can be directly below the portion, or an intervening portion (e.g., layer, structure, region) may also be present. A portion that is “directly adjacent”, “directly on”, “immediately adjacent”, “in contact with”, or “directly supported by” another portion means that no intervening portion is present. It should also be understood that when a portion is referred to as being “on”, “above”, “adjacent”, “over”, “overlying”, “in contact with”, “below”, or “supported by” another portion, it may cover the entire portion or a part of the portion.

[0170] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

1. CLAIMSWhat is claimed is:

1. A composition, comprising: a protein and / or biologic; and a precipitant, wherein the composition comprises greater than or equal to 0.00005 mg / mL and less than or equal to 25 mg / mL of a dehydrating fluid.

2. The composition of claim 1, wherein the composition is in the form of a solid particle.

3. The composition of any one of claims claim 1-2, further comprising a crosslinked polymer.

4. The composition of claim 3, wherein the crosslinked polymer comprises crosslinked polyalkylene oxide, a crosslinked vinylsulfuone / thiol, crosslinked PEG, crosslinked PEGDA, a natural polysaccharide, a modified polysaccharide, and / or alginate.

5. The composition of any one of claims 1-4, wherein the precipitant comprises polyethylene glycol, polyethyleneimine, ammonium sulfate, ethanol, acetone, and / or trichloroacetic acid.

6. The composition of any one of claims 1-5, wherein the precipitant is present in a concentration of greater than or equal to 0.5 wt%.

7. The composition of any one of claims 1-6, wherein the precipitant is present in a concentration of greater than or equal to 5 wt%.

8. The composition of any one of claims 1-7, wherein the dehydrating fluid is a class 3 ICH solvent.

9. The composition of any one of claims 1-8, wherein the dehydrating fluid comprises pentanol, octanol, dodecanol, isopropyl alcohol, ethanol, ethanol, propanol, butanol, 2-methyl-l-proponal, 2-methyl-2-propanol, tert-butanol, 3-methyl-l -butanol, 2,2-dimethyl-l -propanol, hexanol, cyclohexanol, heptanol, octanol, nonanol, decanol, 2- propen-l-ol; benzyl alcohol, phenylmethanol, diphenylmethanol, undecanol, propyldecanol, butadecanol, pentadecanol, hexadecanol, triphenylmethanol, an unsaturated alcohol, and / or a cyclic, olefinic or alkynyl alcohol and / or an alcohol incorporating an internal and / or external heteroatom, and / or an isomer thereof and / or derivative of any of these.

10. The composition of any one of claims 1-9, further comprising a surfactant.

11. The composition of claim 10, wherein the surfactant comprises a polysorbate, a span, a pegylated fatty ester, a pegylated fatty ether, a sucrose ester, a block copolymer, and / or an ethoxylated triglyceride.

12. A suspension, comprising: an aqueous medium; a stabilizing agent contained within the aqueous medium; a plurality of solid particles suspended within the aqueous medium, each particle within the plurality of particles comprising: a protein and / or biologic, a precipitant, and a dehydrating fluid.

13. The suspension of claim 12, wherein the stabilizing agent comprises polyethylene glycol, polyethyleneimine, ammonium sulfate, ethanol, acetone, and / or trichloroacetic acid.

14. The suspension of any one of claims 12-13, wherein the precipitant comprises polyethylene glycol, polyethyleneimine, ammonium sulfate, ethanol, acetone, and / or trichloroacetic acid.

15. The suspension of any one of claims 12-14, wherein the precipitant is present in a concentration of greater than or equal to 0.5 wt%.

16. The suspension of any one of claims 12-15, wherein the precipitant is present in a concentration of greater than or equal to 5 wt%.

17. The suspension of any one of claims 12-16, wherein the dehydrating fluid is a class 3 ICH solvent.

18. The suspension of any one of claims 12-17, wherein the dehydrating fluid comprises pentanol, octanol, dodecanol, isopropyl alcohol, ethanol, ethanol, propanol, butanol, 2-methyl-l-proponal, 2-methyl-2-propanol, tert-butanol, 3-methyl-l -butanol, 2,2-dimethyl-l -propanol, hexanol, cyclohexanol, heptanol, octanol, nonanol, decanol, 2- propen-l-ol; benzyl alcohol, phenylmethanol, diphenylmethanol, undecanol, propyldecanol, butadecanol, pentadecanol, hexadecanol, triphenylmethanol, an unsaturated alcohol, and / or a cyclic, olefinic or alkynyl alcohol and / or an alcohol incorporating an internal and / or external heteroatom, and / or an isomer thereof and / or derivative of any of these.

19. The composition or suspension of any one of claims 12-18, further comprising a surfactant.

20. The composition or suspension of claim 19, wherein the surfactant comprises a polysorbate, a span, a pegylated fatty ester, a pegylated fatty ether, a sucrose ester, a block copolymer, and / or an ethoxylated triglyceride.

21. The suspension of any one of claims 12-20, wherein the stabilizing agent and the precipitant have the same composition.

22. The suspension of any one of claims 12-21, wherein the stabilizing agent and the precipitant have different compositions, and the stabilizing agent comprises polyethylene glycol, polyethyleneimine, ammonium sulfate, ethanol, acetone, and / or trichloroacetic acid.-M -23. The suspension of any one of claims 12-22, wherein the aqueous medium comprises a buffer.

24. The suspension of claim 23, wherein the buffer comprises a HEPES buffer.

25. The suspension of any one of claims 12-24, wherein the solid particles are suspended within the aqueous medium in a concentration of greater than or equal to 350 mg / mL.

26. The suspension of any one of claims 12-25, wherein the solid particles are suspended within the aqueous medium in a concentration of greater than or equal to 400 mg / mL.

27. The suspension of any one of claims 12-26, wherein the solid particles are suspended within the aqueous medium in a concentration of greater than or equal to 450 mg / mL.

28. The suspension of any one of claims 12-27, further comprising a surfactant contained within the aqueous medium.

29. The suspension of any one of claims 12-28, further comprising precipitated biologic and / or protein contained within the aqueous medium.

30. The suspension of 29, wherein greater than or equal to 90% of the precipitated biologic and / or protein comprises monomers of the biologic and / or protein.

31. The composition of any one of claims 12-30, wherein at least a portion of the solid particles in the plurality of solid particles comprises a crosslinked polymer.

32. The composition of claim 31, wherein the crosslinked polymer comprises crosslinked polyalkylene oxide, a crosslinked vinylsulfuone / thiol, crosslinked PEG,crosslinked PEGDA, a natural polysaccharide, a modified polysaccharide, and / or crosslinked alginate.

33. A method, comprising: providing a protein and / or biologic and a precipitant dissolved and / or suspended in a carrier fluid; removing at least some of the carrier fluid to a dehydrating phase; and forming a solid particle comprising the protein and / or biologic.

34. The method of claim 33, wherein the carrier fluid comprises a buffer.

35. The method of claim 34, wherein the buffer comprises a HEPES buffer.

36. The method of any one of claims 33-35, wherein the carrier fluid comprises a salt in a concentration greater than or equal to 100 mM.

37. The method of any one of claims 33-36, wherein the dehydrating phase is a class 3 ICH solvent.

38. The method of any one of claims 33-37, wherein the dehydrating phase comprises pentanol, octanol, dodecanol, isopropyl alcohol, ethanol, ethanol, propanol, butanol, 2-methyl-l-proponal, 2-methyl-2-propanol, tert-butanol, 3-methyl-l -butanol, 2,2-dimethyl-l -propanol, hexanol, cyclohexanol, heptanol, octanol, nonanol, decanol, 2- propen-l-ol; benzyl alcohol, phenylmethanol, diphenylmethanol, undecanol, propyldecanol, butadecanol, pentadecanol, hexadecanol, triphenylmethanol, an unsaturated alcohol, and / or a cyclic, olefinic or alkynyl alcohol and / or an alcohol incorporating an internal and / or external heteroatom, and / or an isomer thereof and / or derivative of any of these.

39. The method of any one of claims 33-38, wherein converting the protein and / or biologic to a solid particle comprises forming droplets comprising the protein and / or biologic and the carrier phase and substantially simultaneously removing at least some ofthe carrier fluid to the dehydrating phase and precipitating the protein and / or biologic within the droplet to form the solid particle.

40. The method of any one of claims 33-39, wherein the carrier fluid comprises a polymer.

41. The method of 40, further comprising cross-linking the polymer within the carrier fluid.

42. The method of claim 41, wherein the cross-linking occurs before and / or during the removing of the carrier fluid and the converting of the protein and / or biologic to a solid particle.

43. The method of claim 41, wherein the cross-linking occurs after the removing of the carrier fluid and the converting of the protein and / or biologic to a solid particle.

44. A method, comprising: providing a protein and / or biologic and a precipitant dissolved and / or suspended in a carrier fluid; in a droplet-forming phase, forming a droplet comprising the carrier fluid, the protein and / or biologic, and the precipitant; removing at least some of the carrier fluid of the droplet to a dehydrating phase; and forming a solid particle comprising the protein and / or biologic.

45. The method of claim 44, wherein the carrier fluid comprises a buffer.

46. The method of claim 45, wherein the buffer comprises a HEPES buffer.

47. The method of any one of claims 44-46, wherein the carrier fluid comprises a salt in a concentration greater than or equal to 100 mM.

48. The method of any one of claims 44-47, wherein the dehydrating phase is a class 3 ICH solvent.

49. The method of any one of claims 44-48, wherein the dehydrating phase comprises pentanol, octanol, dodecanol, isopropyl alcohol, ethanol, ethanol, propanol, butanol, 2-methyl-l-proponal, 2-methyl-2-propanol, tert-butanol, 3-methyl-l -butanol, 2,2-dimethyl-l -propanol, hexanol, cyclohexanol, heptanol, octanol, nonanol, decanol, 2- propen-l-ol; benzyl alcohol, phenylmethanol, diphenylmethanol, undecanol, propyldecanol, butadecanol, pentadecanol, hexadecanol, triphenylmethanol, an unsaturated alcohol, and / or a cyclic, olefinic or alkynyl alcohol and / or an alcohol incorporating an internal and / or external heteroatom, and / or an isomer thereof and / or derivative of any of these.

50. The method of any one of claims 44-49, wherein converting the protein and / or biologic to a solid particle comprises providing the droplets comprising the protein and / or biologic and the carrier phase and substantially simultaneously removing at least some of the carrier fluid to the dehydrating phase and precipitating the protein and / or biologic within the droplet to form the solid particle.

51. The method of any one of claims 44-50, wherein the carrier fluid comprises a polymer.

52. The method of 51, further comprising cross-linking the polymer within the carrier fluid.

53. The method of claim 52, wherein the cross-linking occurs before and / or during the removing of the carrier fluid and the converting of the protein and / or biologic to a solid particle.

54. The method of claim 52, wherein the cross-linking occurs after the removing of the carrier fluid and the converting of the protein and / or biologic to a solid particle.

55. The method of any one of claims 33-54, further comprising suspending the solid particulate in an aqueous phase comprising a precipitant.

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