Hyaluronidase particles, compositions comprising same, and methods of making and using same

Abstract

Described herein are hyaluronidase particles, compositions comprising the same, and methods of making and using the same. The compositions of the invention may comprise hyaluronidase particles and may be a suspension. Granules comprising hyaluronidase may have a moisture content of less than about 15% by weight of the particle and / or a water activity of less than about 0.9. In some embodiments, the methods of the invention may comprise administering to a subject a particle comprising hyaluronidase subcutaneously, intramuscularly, and / or intradermally, where the particle has a moisture content of less than about 15% by weight of the particle and / or a water activity of less than about 0.9.

Description

[0001] Related Application Information This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 486,013, filed February 20, 2023, the disclosure of which is incorporated herein in its entirety by this reference.

[0002] Statement as to Electronic Filing of a Sequence Listing The sequence listing named 1458-5WO_ST26.xml, size 1,557,299 bytes, created on February 19, 2024, and submitted herewith in XML format is hereby incorporated by reference into the specification for all purposes. TECHNICAL FIELD

[0003] The present invention relates to hyaluronidase particles, compositions comprising hyaluronidase particles, and methods of making and using hyaluronidase particles. BACKGROUND

[0004] Subcutaneous injection provides a flexible and effective route of drug administration, in which a drug is injected into the tissue layer between the skin and the muscle. Subcutaneous injection is advantageous because it is a less expensive and less painful parenteral administration method with low risk of systemic infection and slow adsorption rate, which allows long-term action of the injected drug. However, subcutaneously injected fluids remain mostly at the injection site for hours to days before slowly dissipating over the course of several hours to days. Injecting large volumes (e.g., more than about 2 mL) results in fluid retention and / or swelling of the injection area, which can cause local adverse reactions that cause pain, irritation, and / or skin damage. For this reason, subcutaneous injection is generally limited to small doses, e.g., volumes less than about 2 mL.

[0005] Hyaluronidases are a family of enzymes, specifically endoglycosidases, that can break down hyaluronic acid and can be able to degrade other glycosaminoglycans, such as chondroitin and chondroitin sulfate. Hyaluronidases are found in both organs such as testes, spleen, and liver, and body fluids such as tears and blood. There are three different classes of hyaluronidases - mammalian or vertebrate, bacterial, and leech / hookworm. In the mammalian class, the human genome contains six known genes that encode hyaluronidase-like sequences. Purified human, such as testicular hyaluronidase PH-20 or ovine, such as ovine PH-20 hyaluronidase, are commonly used for medical and experimental purposes.

[0006] Hyaluronidase in aqueous solution has been used subcutaneously to alter tissue permeability by degrading the subcutaneous hyaluronic acid network. By breaking down the hyaluronic acid network in the subcutaneous space, subcutaneous administration of hyaluronidase in aqueous solution can allow a subcutaneously injected fluid to dissipate more quickly than without hyaluronidase, which can enable administration of a larger volume of fluid. Hyaluronidase in aqueous solution is approved by the U.S. Food and Drug Administration (FDA) for rapid fluid administration of 1,000 mL or greater in volume, or as an adjuvant to the subcutaneous delivery of other drugs. Hyaluronidase is generally administered as an aqueous solution prior to injection of a large volume dose of greater than 2 mL or as a coformulation with a large volume dose of greater than 2 mL.

[0007] There is a need for new methods for administering hyaluronidase and therapeutics, particularly for subcutaneous and intradermal administration. SUMMARY

[0008] A first aspect of the present invention relates to a particle comprising: a hyaluronidase, wherein the particle has a water content of less than about 15% by weight of the particle and / or a water activity of less than about 0.9.

[0009] A further aspect of the present invention relates to a particle comprising: a hyaluronidase and a therapeutic agent, wherein the particle has a water content of less than about 15% by weight of the particle and / or a water activity of less than about 0.9.

[0010] Another aspect of the present invention relates to a particle comprising: a hyaluronidase and a stabilizer, wherein the particle has a water content of less than about 15% by weight of the particle and / or a water activity of less than about 0.9.

[0011] A further aspect of the present invention relates to a composition comprising a particle as described herein.

[0012] Another aspect of the present invention relates to a composition comprising: a solvent and a particle comprising a hyaluronidase, wherein the particle has a water content of less than about 15% by weight of the particle and / or a water activity of less than about 0.9.

[0013] A further aspect of the present invention relates to a method of increasing the solubility of a therapeutic agent in a liquid, the method comprising: combining the liquid, the therapeutic agent, and a particle comprising a hyaluronidase, wherein the particle has a water content of less than about 15% by weight of the particle and / or a water activity of less than about 0.9, thereby increasing the solubility of the therapeutic agent in the liquid.

[0014] Another aspect of the application relates to a method of improving tolerability and / or increasing absorption of a therapeutic agent upon subcutaneous, intramuscular, and / or intradermal administration to a subject, the method comprising: subcutaneously, intramuscularly, and / or intradermally administering to the subject a particle comprising a hyaluronidase, wherein the particle has a moisture content of less than about 15% by weight of the particle and / or a water activity of less than about 0.9, thereby improving tolerability and / or increasing absorption of the therapeutic agent upon subcutaneous, intramuscular, or intradermal administration to the subject. In some embodiments, the therapeutic agent is subcutaneously, intramuscularly, and / or intradermally administered to the subject before, during, and / or after the particle is subcutaneously, intramuscularly, and / or intradermally administered to the subject.

[0015] Notably, aspects of the application described herein with respect to one embodiment can be incorporated into different embodiments, although not specifically described with respect to the described embodiment. That is, all embodiments and / or features of any embodiment can be combined in any manner and / or combination. Applicant reserves the right to change any originally filed claim, and / or accordingly to submit any new claims, including the right to amend any originally filed claim to depend from any one or more other claims and / or to incorporate any feature of any one or more other claims, although originally disclaimed. These and other objects and / or aspects of the present application are explained in detail in the specification that follows. Those skilled in the art will appreciate the further features, advantages and details of the application upon reading the following detailed description, taken in conjunction with the drawings that illustrate, by way of example, the principles of the application. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is a plot of absorbance at 280 nm over time (hours) showing the amount of bovine gamma globulin (BGG) released from a benzyl benzoate suspension into a medium comprising phosphate buffered saline (PBS) and 0.25 mg / mL hyaluronidase (PBS-Hyal medium) or a medium comprising PBS, 0.5% hyaluronic acid, and 0.25 mg / mL hyaluronidase (PBS-HA-Hyal medium).

[0017] Figure 2 is a plot of absorbance at 280 nm over time (hours) showing the amount of BGG released from a benzyl benzoate suspension into PBS for three independent samples (i.e., PBS medium-1, PBS medium-2, and PBS medium-3).

[0018] Figure 3is a plot of absorbance at 280 nm as a function of time (hours) showing the amount of BGG released from a benzyl benzoate suspension into a medium comprising 0.5% hyaluronic acid (HA) and PBS for three independent samples (i.e., PBS-HA medium-1, PBS-HA medium-2, and PBS HA medium-3).

[0019] Figure 4 is a plot of absorbance at 280 nm as a function of time (hours) showing the amount of BGG released from a benzyl benzoate suspension into PBS for three independent samples (i.e., PBS medium-1, PBS medium-2, and PBS medium-3), the suspension comprising a plurality of particles, each of the particles comprising BGG and a hyaluronidase according to some embodiments of the application.

[0020] Figure 5 is a plot of absorbance at 280 nm as a function of time (hours) showing the amount of BGG released from a benzyl benzoate suspension into a medium comprising 0.5% hyaluronic acid (HA) and PBS for three independent samples (i.e., PBS-HA medium-1, PBS-HA medium-2, and PBS-HA medium-3), the suspension comprising a plurality of particles, each of the particles comprising BGG and a hyaluronidase according to some embodiments of the application.

[0021] Figure 6 is a plot of the stability of microbatches of various hyaluronidase powder formulations stored at 25°C, indicated by enzymatic activity compared to the activity level of the pre-storage stock (i.e., at the time the stock was initially formed, t=0).

[0022] Figure 7 is a plot of the stability of microbatches of various hyaluronidase powder formulations stored at 2-8°C, indicated by enzymatic activity compared to the activity level of the pre-storage stock.

[0023] Figure 8 is a plot of the activity of hyaluronidase as a function of time when stored in a powder formulation containing the enzyme and bovine serum albumin (BSA) at the indicated temperatures.

[0024] Figure 9 is a plot of the change in the percentage of monomers of BSA in a powder containing either just BSA or a combination of BSA and hyaluronidase as a function of time.

[0025] Figure 10 is a microscopy image showing the spherical morphology of particles of a monoclonal antibody (mAb) and ~0.3% hyaluronidase suspended in octanol.

[0026] Figure 11 is a plot of mAb stability as indicated by the percentage of monomer from particles containing only the mAb or the mAb in combination with hyaluronidase when stored at 2-8°C.

[0027] Figure 12 is a plot of mAb stability as indicated by the percentage of high molecular weight species (HMWS) from particles containing only the mAb or the mAb in combination with hyaluronidase when stored at 2-8°C.

[0028] Figure 13 is a plot of mAb stability as indicated by the percentage of monomer from particles containing only the mAb or the mAb in combination with hyaluronidase when stored at 25°C.

[0029] Figure 14 is a plot of mAb stability as indicated by the percentage of HMWS from particles containing only the mAb or the mAb in combination with hyaluronidase when stored at 25°C.

[0030] Figure 15 is a plot of mAb stability as indicated by the percentage of monomer from particles containing only the mAb or the mAb in combination with hyaluronidase when stored at 40°C.

[0031] Figure 16 is a plot of mAb stability as indicated by the percentage of HMWS from particles containing only the mAb or the mAb in combination with hyaluronidase when stored at 40°C.

[0032] Figure 17 is a plot of the activity of hyaluronidase in particles containing only the mAb or the mAb in combination with hyaluronidase over time when stored at 2-8, 25, or 40°C. DETAILED DESCRIPTION

[0033] The present application now being described by reference to the drawings in which embodiments of the application are shown. This application may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and fully convey the scope of the application to those skilled in the art.

[0034] The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in the description of the application and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0035] It will be understood that, although the terms“first,”“second,” etc. can be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. Thus, a“first” element discussed below could also be termed a“second” element without departing from the teachings of the present application. The order of operations (or steps) is not limited to the order shown in a claim or figure unless otherwise explicitly stated.

[0036] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an overly formal or overly literal sense unless expressly so defined herein.

[0037] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict between the specifications and the references, the specifications shall control.

[0038] Also as used herein,“and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0039] Unless otherwise stated, it is specifically intended that the various features of the application described herein can be used in any combination. Moreover, it is intended that the application can be practiced with any feature or combination of features recited herein, unless the context indicates otherwise. For example, if a description states that a complex comprises components A, B, and C, it is specifically intended that A, B, or C, or any combination thereof, can be omitted and / or substituted.

[0040] As used herein, the transitional phrase“consisting essentially of’ (and grammatical variations thereof) is to be interpreted as including those items listed after the phrase“and those that do not materially affect the basic and novel characteristics of the claimed application.” See, In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in original); see also MPEP § 2111.03. Thus, the term“consisting essentially of’ as used herein should not be interpreted as equating to“comprising.”

[0041] It will also be appreciated that, as used herein, the terms "instance", "exemplary", and grammatical variations thereof, are intended to refer to non-limiting examples and / or variant embodiments discussed herein, and are not intended to indicate a preference for one or more embodiments discussed herein over one or more other embodiments.

[0042] The term "about", as used herein when referring to a measurable value such as an amount or concentration and the like, is meant to encompass variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified value. For example, "about X", where X is a measurable value, is meant to include X and variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of X. Ranges of values provided herein can include any other ranges and / or individual values therein.

[0043] Statements regarding ranges of values herein are only intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a range of 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed.

[0044] A“portion” or“fragment” of a material or component, as used herein, refers to less than all (e.g., less than 100%) of the material or component or measurable value thereof. In some embodiments, a“portion” or“fragment” of a material or component refers to about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% (e.g., a portion of length, volume, weight, sequence, etc.) of the material or component or measurable value of the material or component. In some embodiments, a“portion” or“fragment” of a particle or plurality of particles refers to about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the particle or plurality of particles, respectively. In some embodiments, a material or component can be a nucleotide sequence or polypeptide (optionally including a domain), and thus a“portion” or“fragment” of a nucleotide sequence or polypeptide will be understood to mean a nucleotide sequence or polypeptide of reduced length relative to a reference nucleotide sequence or polypeptide (e.g., reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more residues (e.g., one or more nucleotides or one or more peptides), respectively, and comprising, consisting essentially of, and / or consisting of a nucleotide sequence or polypeptide of contiguous residues that are identical or nearly identical (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to the reference nucleotide sequence or polypeptide, respectively. In some embodiments, a“portion” or“fragment” of a nucleotide sequence or polypeptide will have similar or identical biological activity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, or 130% similar biological activity) as compared to a reference nucleotide sequence or polypeptide.In some embodiments, a "portion" or "fragment" of a nucleotide sequence or polypeptide will have reduced biological activity (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% reduced biological activity) or increased biological activity (e.g., 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200%, or more increased biological activity) compared to the reference nucleotide sequence or polypeptide.

[0045] The terms "comprise," "comprises," and "comprising," as used herein, specify the presence of stated features, integers, steps, operations, elements, and / or components but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0046] As used herein, the terms "increase," "increased," "increasing," "enhance," "enhancing," "improve," and "improving" (and grammatical variations thereof) describe an elevation, for example, of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500%, or more, compared to another measurable property or amount (e.g., a control value).

[0047] As used herein, the terms "reduce," "reduced," "reducing," "reduction," "weaken," and "decrease" (and grammatical variations thereof) describe a decrease, for example, of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%, compared to another measurable property or amount (e.g., a control value). In some embodiments, a reduction can result in no or substantially no (i.e., an insignificant amount, e.g., less than about 10% or even 5%) detectable activity or amount.

[0048] According to embodiments of the present application, particles comprising a hyaluronidase are provided. In some embodiments, the particles comprising a hyaluronidase further comprise an excipient (e.g., a stabilizer) and / or a therapeutic agent. The particles of the present application can be solid and can have a moisture content of less than about 15% by weight of the particle and / or a water activity of less than about 0.9. In some embodiments, the particles have a moisture content of less than about 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5% by weight of the particle. In some embodiments, the particles of the present application have a moisture content ranging from about 0%, 0.5%, 1%, 2%, 3%, or 4% to about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% by weight of the particle. In some embodiments, the particles of the present application have a moisture content of less than about 10% by weight of the particle or less than about 5% by weight of the particle. In some embodiments, the particles of the present application have a moisture content of about 0%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% by weight of the particle. In some embodiments, the particles of the present application have a water activity of less than about 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, 0.05, or 0.01. In some embodiments, the particles of the present application have a water activity of less than about 0.5 or less than about 0.1. In some embodiments, the particles have a water activity of from about 0, 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, or 0.45 to about 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, or 0.9.

[0049] Water content can be measured using methods known in the art, for example, by using Karl Fischer titration and / or thermogravimetric analysis. In some embodiments, the water content of the particles of the application can be measured using a Mettler Toledo coulometric KF titrator. A sample comprising the particles of the application can be contacted with a solvent (e.g., methanol) to extract water from the sample, and then the amount of water in the solvent is measured (e.g., using a Mettler Toledo coulometric KF titrator), and the amount of water present in the sample is calculated from the water content of the solvent after subtracting any water present in the solvent blank (solvent before contact with the particles). For example, in some embodiments, about 15 mg to about 30 mg of the particles of the application (e.g., in the form of a powder) or a composition (e.g., a suspension) comprising the particles of the application is weighed into a glass vial in a dry box (relative humidity (RH) less than 20%) and about 1.5 mL of anhydrous methanol is added to the particles or composition to extract water from the sample. The methanol can then be injected into a titration cell (e.g., of a Mettler Toledo coulometric KF titrator), and the amount of water present in the methanol can be measured. The amount of water present in the particles or composition can then be calculated from the water content of the methanol after subtracting any water present in the methanol blank. In some embodiments, the water content of a suspension can be measured by injecting the suspension directly into a titration cell. Water content can also be measured by loss on drying (e.g., using thermogravimetric analysis (TGA)), for example, when there are no volatile components present in the sample or no components in the sample that will evaporate at temperatures similar to water.

[0050] Water activity is a measure of the chemical availability of water in a sample (e.g., in a particle of the application), and can quantify the amount of unbound water in a sample and / or how tightly water is bound to components in the sample (e.g., how tightly water is bound to a biologic present in a particle). In some embodiments, water activity is measured by equilibrating a sample comprising a particle of the application in a closed chamber and measuring the relative humidity of the headspace in the chamber. Alternatively or additionally, water activity can be measured by correlating water content and water activity in a known system. To correlate water content and water activity, the water activity of a sample comprising a particle of the application is determined by equilibrating the sample with a vapor phase having a known water activity (e.g., a saturated salt solution or using a dynamic vapor sorption instrument), and measuring the change in water content or measuring the change in mass by Karl Fischer titration to calculate the water activity of the sample. Thus, the water content and / or mass of the sample can be measured before and after contact with the vapor phase, and the change used to measure the water activity of the sample. In some embodiments, the water content and water activity of an individual particle of the application or a droplet comprising a particle of the application can be correlated by measuring the change in volume of the individual particle or droplet upon contact (e.g., exposure to) a solvent having a known water activity or a gas having a known water activity, as described in Rickard et al., Biophysical Journal, 2010; 98(6): 1075-84. The volume of the individual particle or droplet can be measured before and after contact with the solvent or gas, and the change in volume used to measure the water activity of the sample.

[0051] In some embodiments, a particle of the application has a water content (e.g., total moisture content) of less than about 3% of the total particle mass. A particle can have a total moisture content of less than about 2.5%, 2%, 1.5%, 1%, or 0.5% of the total particle mass. In some embodiments, the total moisture content of a particle is in the range of about 0%, 0.1%, or 0.5% to about 1%, 1.5%, 2%, 2.5%, or 3% of the total particle mass.

[0052] The particles of the present application can have a residual content in an amount of less than about 3% by weight of the particles (e.g., total particle mass). The particles can have a residual content of less than about 2.5%, 2%, 1.5%, 1%, or 0.5% by weight of the particles. In some embodiments, the particles of the present application have a residual content in a range of about 0%, 0.01%, 0.05%, 0.1%, or 0.5% to about 1%, 1.5%, 2%, 2.5%, or 3% by weight of the particles. In some embodiments, the particles of the present application have a total residual content (e.g., sum of all residuals) of less than 3% by weight of the particles, for example, in an amount of less than 2.5%, 2%, 1.5%, 1%, or 0.5% by weight of the particles. In some embodiments, an individual residual can be present in the particles of the present application in an amount of less than 1% by weight of the particles, for example, in an amount of about 0.01%, 0.05%, 0.1%, or 0.2% to about 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, or 0.9% by weight of the particles. In some embodiments, an individual residual can be present in the particles of the present application in an amount of less than 0.5% by weight of the particles, for example, in an amount of about 0.01%, 0.05%, 0.1%, 0.2% to 0.3%, or 0.4% by weight of the particles. The residual can be one or more components present in a composition used to make (e.g., form, dehydrate, solidify, wash, and / or isolate) the particles of the present application. Exemplary residuals include, but are not limited to, solvents (e.g., octanol and / or amyl alcohol). In some embodiments, the residual can be a Class II or Class III residual solvent as classified by the U.S. Food and Drug Administration.

[0053] The particles of the present application can be microparticles or nanoparticles. In some embodiments, the particles can have a size and / or diameter in at least one dimension in the range of about 1, 5, 10, 20, 30, 40, or 50 nm to about 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nm. In some embodiments, the plurality of particles of the present application have an average size of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nm. In some embodiments, the particles of the present application can have a size and / or diameter in at least one dimension in the range of about 0.1, 0.5, 1, 5, 10, or 15 microns to about 20, 25, or 30 microns. In some embodiments, the particles can have a size and / or diameter in at least one dimension of about 0.1, 0.5, 1, or 2 microns to about 3, 4, 5, 6, 7, 8, 9, or 10 microns. In some embodiments, the particles can have a size and / or diameter in at least one dimension of about 5, 6, 7, 8, 9, or 10 microns to about 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 microns. In some embodiments, the particles can have a size and / or diameter in at least one dimension of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 microns to about 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 microns.

[0054] The particles of the present application can be spherical in shape. In some embodiments, the particles can be amorphous. In some embodiments, at least a portion of the particles are amorphous, thus the particles can be partially amorphous. For example, all or a portion of the hyaluronidase in the particles of the present application can be amorphous, but molecules (e.g., salts) within the matrix of the hyaluronidase in the particles of the present application can be crystalline. The particles of the present application can comprise one or more hyaluronidase molecules that are the same or different from each other and / or one or more matrices of hyaluronidase molecules that are the same or different from each other, optionally wherein the hyaluronidase in the particles is amorphous.

[0055] In some embodiments, the particles of the application are in solid form, e.g., solid particulates and / or powders. In some embodiments, the particles of the application are in solid form when the particles of the application are in powder form (e.g., dry powder, such as a powder without a liquid dispersed or suspended in the powder) and / or when the particles are present in a vehicle (e.g., a non-aqueous solvent and / or an aqueous solvent). In some embodiments, the particles of the application are in the form of a powder. In some embodiments, the particles of the application can be microglassified particles and / or the hyaluronidase can be in the form of a microglassified hyaluronidase as a particle and / or contained in a particle. As used herein, "microglassification" refers to a process of removing water (e.g., dehydrating) from a component (e.g., a hyaluronidase or a therapeutic agent) present in (e.g., dissolved or suspended in) an aqueous composition by contacting the aqueous composition with an organic phase (e.g., a non-aqueous organic phase) to provide a solid comprising the component, and the solidified component can be referred to herein as a "microglassified" component. The microglassification process can be performed in any manner that allows the organic phase and the aqueous composition comprising the component to contact so as to remove water from the component. For example, in some embodiments, a homogenizer (e.g., an inline homogenizer) and / or a microfluidic device can be used to contact the organic phase and the aqueous composition comprising the component, and / or the microglassification process can be performed by spraying droplets of the aqueous composition comprising the component into the organic phase or vice versa so as to contact the organic phase and the aqueous composition. In the microglassification process, the organic phase is a liquid organic phase (e.g., a liquid dehydrating composition), and the aqueous composition is a liquid. Thus, the microglassification process includes a liquid-liquid mixture and / or system. The microglassification process can include contacting at least two different liquids. In some embodiments, the microglassification process does not involve a spray-drying step or method and / or does not involve spraying droplets of the aqueous composition comprising the component into the organic phase or vice versa. In some embodiments, the microglassification process without a spray-drying step or method does not involve spraying droplets of the aqueous composition comprising the component into a gas (e.g., a heated gas phase) and / or the microglassification process does not have a liquid-gas mixture and / or system. The microglassified component is not a precipitate. In some embodiments, the particles of the application are not prepared by a method that uses a phase separation agent. As used herein, a "phase separation agent" refers to an agent that causes a biological agent to precipitate from a solution or that causes a liquid-liquid phase separation, where one of the liquid phases comprises a majority of the biological agent. Exemplary phase separation agents include, but are not limited to, salts (e.g., those useful for "salting out proteins") and polyethylene glycols (PEGs). The phase separation as a process of nucleation and growth can occur during a microglassification method (e.g., liquid-liquid and / or liquid-solid), but the microglassified component is not formed by phase separation.In contrast, microglassification is a continuous phase change that does not require a nucleation event to form a solidified component. Microglassified components (e.g., microglassified hyaluronidase) can dissolve back into the original composition (e.g., aqueous solution) and / or aqueous composition (e.g., aqueous buffer) in which they were located.

[0056] In some embodiments, microglassified components (e.g., microglassified hyaluronidase) can be more stable (e.g., have increased duration of shelf life, increased range of acceptable storage temperatures, and / or optionally decreased degradation after repeated freeze-thaw cycles) than solutions of the component (e.g., hyaluronidase solution, where the hyaluronidase has not been microglassified). In some embodiments, microglassification of a component (e.g., hyaluronidase) increases the stability of the component, optionally as compared to the stability of the same component that is not microglassified, as present in solution. In some embodiments, particles comprising hyaluronidase of the present application have increased stability as compared to hyaluronidase in solution, where the hyaluronidase in solution is not in particle form and has not been microglassified.

[0057] Particles comprising hyaluronidase can exist in any form, such as, for example, microparticles, nanoparticles, microspheres, or nanospheres. Particles of the present application can be amorphous and / or crystalline. In some embodiments, a plurality of particles of the present application comprises amorphous particles. In some embodiments, a plurality of particles of the present application comprises crystalline particles, such as, for example, when a small molecule (e.g., a salt) that can crystallize is present. A plurality of particles of the present application can be uniform in size, or can be polydisperse. In some embodiments, at least a portion of the plurality of particles have a size within about ±5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, or more of the average particle size. In some embodiments, a composition of the present application comprises discrete particles. Particles of the present application can have a density of about 0.5, 0.6, 0.7, 0.8, 0.9, or 1 g / cm3to about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 g / cm3. 3 In some embodiments, particles of the present application can have a density of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 g / cm3. 3 In some embodiments, particles of the present application can have a density of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 g / cm3. 3 In some embodiments, particles of the present application can have a density of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 g / cm3. 3 In some embodiments, particles of the present application can have a density of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 g / cm3. 3 Density can be measured and / or determined using methods known in the art, such as, but not limited to, using a gas pycnometer and / or by sedimentation experiments.

[0058] As used herein, " hyaluronidase " refers to the enzyme or its part (for example, its domain) of the glycosidic bond of cutting glycosaminoglycan such as but not limited to hyaluronic acid. As used herein, " hyaluronidase " refers to all forms of hyaluronidase from any organism (for example, from antibacterial and / or mammal, such as human and / or cattle), and naturally occurring (for example, wild-type hyaluronidase) and recombinant hyaluronidase. In some embodiments, the hyaluronidase present in the granule of the present invention at least cuts hyaluronic acid, such as optionally present in the skin of subject and / or the people's hyaluronic acid in tissue. The hyaluronidase of the present invention can cut the β 1-4 key (for example, hyaluronic acid β 1-4 key) of glycosaminoglycan. Exemplary hyaluronidase includes, but is not limited to, human hyaluronidase (for example, people Hyal-1, people Hyal-2, people Hyal-3, people Hyal-4 and / or people PH-20 / Spam1), bovine testis hyaluronidase and / or sheep hyaluronidase (for example, sheep PH-20). For example, in some embodiments, hyaluronidase can be sheep testicle hyaluronidase V (for example, Millipore Sigma catalog number: H6254), optionally with a specific activity of ≥1,500 units / mg solid. The hyaluronidase present in the granules of the present invention can be synthetically obtained (for example, synthesized in a laboratory) and / or obtained and / or derived from nature (for example, from living or previously living organisms). In some embodiments, hyaluronidase can be identical with the hyaluronidase found in nature (that is, natural hyaluronidase) or can be modified from the hyaluronidase found in nature (for example, modified hyaluronidase and / or recombinant hyaluronidase). For example, the hyaluronidase of the present invention can be modified to have a structure, sequence, charge, chemical modification and / or length different from natural hyaluronidase, and / or the hyaluronidase can be a part of natural hyaluronidase (for example, the active domain of natural hyaluronidase). In some embodiments, the hyaluronidase of the present invention can be a commercially available hyaluronidase and / or can have similar activity to a commercially available hyaluronidase (e.g., hyaluronidase such as, but not limited to, (recombinant human hyaluronidase), ALT-BC4 (recombinant human hyaluronidase), ALT-B4 (recombinant human hyaluronidase), MK-5180 (recombinant human hyaluronidase), (recombinant human hyaluronidase; SEQ ID NO: 1), (bovine testicular hyaluronidase; SEQ ID NO: 2), HYDASE TM (bovine testicular hyaluronidase; SEQ ID NO: 2), (sheep testicular hyaluronidase; optionally having the sequence of SEQ ID NO: 3 and / or SEQ ID NO: 4), Vespula vulgaris hyaluronidase, e.g., having the sequence of one of SEQ ID NOs: 21 or 22, Apis mellifera hyaluronidase, e.g., having the sequence of SEQ ID NO: 23, Dolichovespula maculata hyaluronidase, e.g., having the sequence of SEQ ID NO: 24, Polistes annularis hyaluronidase, e.g., having the sequence of SEQ ID NO: 25, Mus musculus hyaluronidase, e.g., having the sequence of any one of SEQ ID NOs: 26-28 or 39, Sus scrofa hyaluronidase, e.g., having the sequence of any one of SEQ ID NOs: 29 or 30, Rattus norvegicus hyaluronidase, e.g., having the sequence of any one of SEQ ID NOs: 31-33 or 38, Oryctolagus cuniculus hyaluronidase, e.g., having the sequence of SEQ ID NO: 34, Pongo pygmaeus hyaluronidase, e.g., having the sequence of SEQ ID NO: 35, Macaca fascicularis hyaluronidase, e.g., having the sequence of SEQ ID NO: 36, Cavia porcellus hyaluronidase, e.g., having the sequence of SEQ ID NO: 37, Staphylococcus aureus hyaluronidase, e.g., having the sequence of SEQ ID NO: 40, Staphylococcus pyogenes hyaluronidase, e.g., having the sequence of SEQ ID NO: 41, Clostridium perfringens hyaluronidase, e.g., having the sequence of SEQ ID NO: 42, and / or a hyaluronidase according to any one of SEQ ID NOs: 5-20 or 43-896).In some embodiments, the hyaluronidase of the present application is a human hyaluronidase, a bovine hyaluronidase (e.g., a bovine testicular hyaluronidase), a sheep hyaluronidase (e.g., a sheep testicular hyaluronidase), a P. vulgaris hyaluronidase, a honeybee hyaluronidase, a P. barbatus hyaluronidase, a P. papatua hyaluronidase, a Mus musculus hyaluronidase, a Sus scrofa hyaluronidase, a Rattus norvegicus hyaluronidase, a Oryctolagus cuniculus hyaluronidase, a Pongo pygmaeus hyaluronidase, a Macaca fascicularis hyaluronidase, a Cavia porcellus hyaluronidase, a Staphylococcus aureus hyaluronidase, a Staphylococcus pyogenes hyaluronidase, a Clostridium perfringens hyaluronidase, and / or a recombinant hyaluronidase of any of the foregoing. In some embodiments, the hyaluronidase of the present application has an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of SEQ ID NOs: 1-896. In some embodiments, the hyaluronidase of the present application comprises the amino acid sequence of one of SEQ ID NOs: 1-896. In some embodiments, the hyaluronidase can be as described in U.S. Patent No. 8,431,380; U.S. Patent No. 10,857,213; U.S. Patent No. 9,284,543; U.S. Patent No. 9,447,401; U.S. Patent Publication No. 2021 / 0155913; U.S. Patent Publication No. US2023 / 0250408; and Stern, R. and Jedrzejas, M. J., Hyaluronidases: Their Genomics, Structures, and Mechanisms of Action. Chem Rev. (2006), 818-839, the contents of each of which are incorporated herein by reference in their entirety.

[0059] Hyaluronidase can be present in the granules of the present application in an amount of about 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% by weight of the granule. In some embodiments, hyaluronidase is present in the granules of the present application in an amount of about 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% to about 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% by weight of the granule. In some embodiments, hyaluronidase is present in the granules of the present application in an amount of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, or 1% to about 2%, 3%, 4%, or 5% by weight of the granule. In some embodiments, hyaluronidase is present in the granules of the present application in an amount of about 50%, 55%, 60%, 65%, or 70% to about 75%, 80%, 85%, 90%, 95%, 99%, or 100% by weight of the granule.

[0060] The particles of the present application and / or the hyaluronidase and / or therapeutic agent present in the particles can dissolve in an aqueous composition upon and / or after contact with the aqueous composition. In some embodiments, contacting the particles of the present application with an aqueous composition can be performed ex vivo and / or in vitro. In some embodiments, contacting the particles of the present application with an aqueous composition can be performed in vivo, for example, during and / or after administration (e.g., subcutaneous administration) to a subject by contacting the particles with a bodily fluid. All or a portion of the particles can dissolve in the aqueous composition and / or all or a portion of the hyaluronidase and / or another component (e.g., a therapeutic agent) present in the particles can dissolve in the aqueous composition. In some embodiments, the particles of the present application and / or components thereof (e.g., hyaluronidase and / or therapeutic agent) can dissolve upon contact with an aqueous composition to a period of time from about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours or more after contact with the aqueous composition, optionally at room temperature (e.g., about 20 °C to about 22 °C) and / or body temperature (e.g., about 33 °C, 34 °C, 35 °C, or 36 °C to about 38 °C). In some embodiments, the particles of the present application and / or components thereof (e.g., hyaluronidase and / or therapeutic agent) dissolve in an aqueous composition within about 15, 30, or 45 seconds, or about 1, 5, 15, 30, or 45 minutes, or about 1, 1.25, 1.5, 1.75, or 2 hours after contact of the particles with the aqueous composition, optionally at a temperature in the range of about 20 °C to about 40 °C. Exemplary aqueous compositions include, but are not limited to, water, saline, a buffer (e.g., phosphate buffered saline), and / or a bodily fluid (e.g., blood and / or interstitial fluid).

[0061] The particles of the present application can maintain and / or preserve one or more properties and / or functions of the hyaluronidase and / or therapeutic agent present in the particles as compared to the same one or more properties and / or functions of the hyaluronidase and / or therapeutic agent prior to formation of the particles and / or as compared to the same one or more properties and / or functions of a control hyaluronidase (e.g., free hyaluronidase) and / or control therapeutic agent that is not provided in the form of a particle. In some embodiments, the particles of the present application can increase the stability of the hyaluronidase and / or therapeutic agent present in the particles as compared to the stability of the hyaluronidase and / or therapeutic agent prior to formation of the particles. The control hyaluronidase can be the same hyaluronidase used in the particles of the present application and / or analogous thereto, and the control therapeutic agent can be the same therapeutic agent used in the particles of the present application and / or analogous thereto. In some embodiments, the control hyaluronidase is a commercially available hyaluronidase that has not yet been provided in the form of a particle of the present application, and the control hyaluronidase can be the same type as the hyaluronidase used in the particles of the present application.

[0062] In some embodiments, after the particles of the application and / or components thereof (e.g., hyaluronidase and / or therapeutic agent) are dissolved (e.g., in whole or in part) in an aqueous composition, the activity of the component (e.g., hyaluronidase and / or therapeutic agent) is within about ±5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the activity of a control component and / or the activity of the component prior to formation in the particles of the application. The activity of the component (e.g., hyaluronidase and / or therapeutic agent) can be measured in the aqueous composition in which the particles are dissolved. The activity of the component (e.g., enzymatic activity of the hyaluronidase) can be measured by methods known in the art. For example, the activity of the hyaluronidase can be measured using the USP XXII Hyaluronidase Assay, EC 3.2.1.35, HAse and / or USP 29-NF 24, page 1057. In some embodiments, the activity of the hyaluronidase and / or changes in the activity of the hyaluronidase can be determined and / or measured by measuring the viscosity of a composition comprising the hyaluronidase and / or by comparing the viscosity of a composition comprising the hyaluronidase after a certain amount of time in contact with hyaluronic acid. For example, in some embodiments, the viscosity of a composition comprising the hyaluronidase can be measured and can be compared to a standard curve prepared using the viscosities of a composition comprising a particular amount of hyaluronidase after two or more different time periods in contact with hyaluronic acid. In some embodiments, the control component is a control hyaluronidase and / or a control therapeutic agent. In some embodiments, the activity of the hyaluronidase can be measured using a turbidimetric assay, such as that described in Dorfman, A. (1955) Methods in Enzymology, Vol. I, 166-173 (one unit equals one NF unit as determined by the method described in USP XXII-NF XVII Joint Edition, page 644 (1990)). For example, in some embodiments, the turbidity (e.g., optical density) of a composition comprising the hyaluronidase can be measured as the percent transmittance of 600 nm light through 1 cm of the hyaluronic acid composition (e.g., a composition (e.g., a solution) comprising about 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, or about 0.5 mg / ml of hyaluronic acid present in a phosphate buffered saline (e.g., a solution)).In some embodiments, a reaction mixture comprising or consisting of 160 mM sodium phosphate, 39 mM sodium chloride, 0.005% (w / v) bovine serum albumin, 0.015% (w / v) hyaluronan, and 1.5-5 units of hyaluronidase is incubated at 37°C for 45 minutes, and then 0.5 mL of the reaction mixture is added to 2.5 mL of an acidic albumin solution (0.1% (w / v) bovine serum albumin pH 3.75, containing 1:1 HC1:H20) at 37°C and incubated for 10 minutes at room temperature. The percent transmittance at 600 nm is measured and compared to a standard curve. In some embodiments, one unit of hyaluronidase activity is equal to a change in A600 of 0.330 per minute at pH 5.35 at 37°C in 2.0 ml of reaction mixture (e.g., a reaction mixture comprising or consisting of 160 mM sodium phosphate, 39 mM sodium chloride, 0.005% (w / v) bovine serum albumin, 0.015% (w / v) hyaluronan, and 1.5-5 units of hyaluronidase). In some embodiments, the activity of a solubilized hyaluronidase and / or therapeutic agent is compared to the activity of the hyaluronidase and / or therapeutic agent prior to formation in a particle of the application. In some embodiments, after a particle of the application is solubilized in an aqueous composition, the enzymatic activity of the hyaluronidase present in the particle is within about ±5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the enzymatic activity of a control hyaluronidase (e.g., free hyaluronidase) and / or the enzymatic activity of the same hyaluronidase prior to formation in a particle of the application. For example, a hyaluronidase having a specific activity of 1,500 units / mg solids (where the solids are in the form of the hyaluronidase prior to formation in a particle (e.g., a lyophilized powder)) can be in and / or used to form a particle of the application (e.g., the particle comprises 1,500 hyaluronidase activity units per mg solids, where the solids are the lyophilized powder of hyaluronidase prior to formation of the particle), and upon solubilization (e.g., in whole or in part) of the particle comprising the hyaluronidase in an aqueous composition, the solubilized hyaluronidase can have a specific activity of at least about 750 units / mg solids. As used herein, "free hyaluronidase" refers to hyaluronidase that has not yet been in a particle of the application.

[0063] In some embodiments, the activity of a component (e.g., a hyaluronidase and / or a therapeutic agent) present within a particle of the present application can be within about ±5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the activity of a control component (e.g., a control hyaluronidase and / or a control therapeutic agent), and / or the activity of the component prior to formation in a particle of the present application, after the particle of the present application is stored and then dissolved (e.g., in whole or in part) in an aqueous composition. In some embodiments, the enzymatic activity of a hyaluronidase present in a particle of the present application can be within about ±5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the enzymatic activity of a control hyaluronidase, and / or the enzymatic activity of the hyaluronidase prior to formation in a particle, after the particle of the present application is stored and then dissolved (e.g., in whole or in part) in an aqueous composition.

[0064] In some embodiments, the activity of a component (e.g., a hyaluronidase and / or a therapeutic agent) present within a particle of the present application can be within about ±5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the activity of a control component (e.g., a control hyaluronidase and / or a control therapeutic agent), and / or the activity of the component prior to formation in a particle of the present application, after the particle of the present application is stored and then dissolved (e.g., in whole or in part) in an aqueous composition. In some embodiments, the enzymatic activity of a hyaluronidase present in a particle of the present application can be within about ±5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the enzymatic activity of a control hyaluronidase, and / or the enzymatic activity of the hyaluronidase prior to formation in a particle, after the particle of the present application is stored and then dissolved (e.g., in whole or in part) in an aqueous composition.

[0065] The particles of the present application can be stored at a temperature of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 °C to about 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 °C for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks, months, and / or years. In some embodiments, the particles of the present application are stored at a temperature of about 2 °C to about 8 or 10 °C for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months. In some embodiments, the particles of the present application are stored at a temperature of about 25 °C or about 20 °C to about 22 °C for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months. In some embodiments, the particles of the present application are stored at a temperature of about 40 °C for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months.

[0066] The therapeutic agent may be present in the particles of the present invention and / or in the compositions of the present invention. In some embodiments, the therapeutic agent may be an agent that can be dehydrated to provide the particles of the present invention and that can provide a therapeutic benefit to the subject. In some embodiments, the therapeutic agent may be an agent that can be microvitrified and that can provide a therapeutic benefit to the subject. In some embodiments, the therapeutic agent is hydrophilic. In some embodiments, the therapeutic agent is hydrophobic. In some embodiments, the therapeutic agent is hydrophobic and is dissolved in a vehicle (e.g., a non-aqueous solvent and / or an aqueous solvent). In some embodiments, the therapeutic agent is a biologic, such as, but not limited to, an amino acid, a peptide, a protein, an enzyme or a fragment thereof, an antibody or a fragment thereof (e.g., a heavy chain, a light chain, a fusion protein, an Fv and / or an Fc), a nucleotide and / or a polynucleotide, such as, for example, an oligonucleotide, a DNA and / or an RNA. Exemplary antibody therapeutics include, but are not limited to, immunoglobulin G (IgG; human IgG), [fam]-trastuzumab deruxtecan, abagovomab, abciximab, adalimumab, ado-trastuzumab emtansine, aducanumab, alemtuzumab, alirocumab, amivantamab, odesivimab-ebgn, anifrolumab, ansuvimab, atezolizumab, atoltivimab, avelumab, axatilimab, bamlavivimab, and sirocumab. mab), basiliximab, bebtelovimab, bedinvetmab, belantamabmafodotin, belimumab, benralizumab, bevacizumab, bezlotoxumab, bimekizumab, blinatumomab, brentuximabvedotin), brodalumab, brolucizumab, burosumab, camrelizumab, canakinumab, caplacizumab, casirivimab, catumaxomab, cemiplimab, certolizumab pegol, cetuximab, cilgavimab, concizumab, cosibelimab, crizanlizumab, crovalimab, daclizumab, daratumumab, denosumab, dinutuximab, dinutuximab beta, donanemab, dostarlimab, dupilumab, durvalumab, eculizumab, edrecolomab, efalizumab, elotuzumab, elranatamab, emapalumab, emicizumab, enfortumab vedotin, epcoritamab, eptinezumab, erenumab, ertumaxomab, etesevimab, evinacumab, evolocumab, faricimab, fremanezumab, frunevetmab, galcanezumab, garadacimab, gemtuzumab, gemtuzumab ozogamicin, girentuximab, glembatumumab, ibalizumab, ibritumomab, icrucumab, idarucizumab, imgatuzumab, inebolizumab, inotuzumab, ipilimumab, iratumumab, ishatsuu, ishatsuu-tnfr2g, ishatsuu-tnfrsfmg, ishatsuu-tnfsig, ishatsuu-tnfsig2, ishatsuu-tnfsig3, ishatsuu-tnfsig4, ishatsuu-tnfsig5, ishatsuu-tnfsig6, ishatsuu-tnfsig7, ishatsuu-tnfsig8, ishatsuu-tnfsig9, ishatsuu-tnfsig10, ishatsuu-tnfsig11, ishatsuu-tnfsig12, ishatsuu-tnfsig13, ishatsuu-tnfsig14, ishatsuu-tnfsig15, ishatsuu-tnfsig16, ishatsuu-tnfsig17, ishatsuu-tnfsig18, ishatsuu-tnfsig19, ishatsuu-tnfsig20, ishatsuu-tnfsig21, ishatsuu-tnfsig22, ishatsuu-tnfsig23, ishatsuu-tnfsig24, ishatsuu-tnfsig25, ishatsuu-tnfsig26, ishatsuu-tnfsig27, ishatsuu-tnfsig28, ishatsuu-tnfsig29, ishatsuu-tnfsig30, ishatsuu-tnfsig31, ishatsuu-tnfsig32, ishatsuu-tnfsig33, ishatsuu-tnfsig34, ishatsuu-tnfsig35, ishatsuu-tnfsig36, ishatsuu-tnfsig37, ishatsuu-tnfsig38, ishatsuu-tnfsig39, ishatsuu-tnfsig40,ozogamicin), glofitamab, golimumab, guselkumab, ibalizumab, ibritumomabtiuxetan, idarucizumab, imdevimab, inebilizumab, infliximab, inotuzumab, and inotuzumab ozogamicin, ipilimumab, isatuximab, itolizumab, ixekizumab, lanadelumab, lebrikizumab, lecanemab, lokivetmab, loncastuximab tesirine, maftivimab, margetuximab, marstacimab, mepolizumab, mirikizumab, mirvetuximab soravtansine, mogamulizumab, mosunetuzumab, moxetumomab pasudotox), muromonab-cd3, narsoplimab, natalizumab, naxitamab, nebacumab, necitumumab, nimotuzumab, nirsevimab, nivolumab, obiltoxaximab, obinutuzumab, ocrelizumab, odronextamab, ofatumumab, olaratumab, omalizumab, palivizumab, panitumumab, patritumabderuxtecan), pembrolizumab, pertuzumab, polatuzumab vedotin, pozelimab, racotumomab, ramucirumab, ranibizumab, ravulizumab, raxibacumab, regdanvimab, relatlimab, reslizumab, retifanlimab, risankizumab, rituximab, romosozumab, rozanolixizumab, ruplizumab, sacituzumab govitecan, sarilumab, satralizumab, secukinumab, serplulimab, siltuximab, sintilimab, sotrovimab, spesolimab, sugemalimab, sutimlimab, tafasitamab, talquetamab, tarlatamab, tebentafusp, teclistamab, teplizumab, tetraptumumab, tezepelumab, tildrakizumab, tislelizumab, tisotumabvedotin), tixagevimab, tocilizumab, toripalimab, tositumomab, tralokinumab, trastuzumab, tremelimumab, ublituximab, ustekinumab, vedolizumab, veltuzumab and zolbetuximab, and fragments of any of the foregoing.

[0067] In some embodiments, the therapeutic agent is IgG (human IgG), abavolumab, adalimumab, alirocumab, betinvetumab, belimumab, benralizumab, bicizumab, bodalumab, brosuzumab, canakinumab, casprevirumab, becelizumab, consizumab, kovalizumab, daclizumab, denosumab, dupilumab, efalizumab, enatuzumab, emicizumab, icaritumomab, eprezumab, evolocumab, ramanezumab, fulvelumab, gacardinib, Nepetuzumab, gadacizumab, guselkumab, ixekizumab, ranarumab, nerezumab, lokivituzumab, matacizumab, mepolizumab, migizone, ofatumumab, omalizumab, parezolizumab, risankizumab, romotuzumab, lorixizumab, salizumab, satrelizumab, secukinumab, taquituzumab, terituzumab, tezerumab, tiriquizumab, tocilizumab, trorocirumab, ustekinumab, veltuzumab, a fragment of any of the foregoing, or a combination of any of them. In some embodiments, the antibody therapeutic is IgG (human IgG), abavolumab, alemtuzumab, daratumumab, denosumab, pertuzumab, rituximab, trastuzumab, veltuzumab, a fragment of any of the foregoing, or a combination of any of them.

[0068] In some embodiments, the therapeutic agent is an antibody conjugated (e.g., covalently bound) to another moiety (e.g., a second therapeutic agent), such as, but not limited to, an antibody drug conjugate (ADC) and / or an antibody oligonucleotide conjugate (AOC). In some embodiments, the therapeutic agent includes an antibody conjugated (e.g., covalently bound) to a tubulin inhibitor (e.g., auristatin, maytansinoid, and / or tubulysin), a DNA damaging agent (e.g., calicheamicin, duocarmycin, exatecan, deruxtecan, govitecan, and / or a pyrrolobenzodiazepine). ) and / or an immunomodulator (e.g., a toll-like receptor agonist and / or stimulator of interferon gene agonist) conjugated antibody.

[0069] In some embodiments, the therapeutic agent is a small molecule having a molecular weight of less than 500 daltons, optionally a small organic molecule (i.e., an organic compound having a molecular weight of less than 500 daltons). Exemplary therapeutic agents include, but are not limited to, antimicrobial agents, anti-inflammatory agents, analgesic agents, anesthetic agents, antihistamine agents, antiseptic agents, immunosuppressive agents, anti-hemorrhagic agents, vasodilators, wound healing agents, antineoplastic agents, antacids, anxiolytic agents, antiarrhythmic agents, antibacterial agents, antibiotics, anticoagulants, thrombolytic agents, anticonvulsants, antidepressants, antidiarrheal agents, antiemetics, antifungal agents, antihypertensive agents, antipsychotic agents, antipyretics, antiviral agents, barbiturates, beta blockers, bronchodilators, cold relief agents, corticosteroids, cough suppressants, cytotoxins, decongestants, diuretics, expectorants, hormones, hypoglycemic agents, laxatives, muscle relaxants, sedatives, sex hormones (female and / or male), sleeping drugs, tranquilizers, vitamins, and / or anti-biofilm agents. In some embodiments, the therapeutic agent is an anti-inflammatory agent, e.g., a corticosteroid. In some embodiments, the therapeutic agent is an anesthetic agent, e.g., bupivacaine. In some embodiments, the therapeutic agent is a hormone, e.g., testosterone (e.g., testosterone enanthate), dihydrotestosterone, androstenedione, estrone, estradiol, estriol, estetrol, estrogens, and / or progesterone. In some embodiments, the hormone therapeutic agent is dissolved in a non-aqueous vehicle (e.g., an oil, e.g., sesame oil), optionally including a particle of the present application. In some embodiments, the therapeutic agent is (e.g., testosterone enanthate). In some embodiments, the composition of the present application includes a non-aqueous vehicle (e.g., an oil, e.g., sesame oil), optionally including a particle of the present application, and and optionally a particle of the present application. One or more (e.g., 1, 2, 3, 4, 5, or more) different therapeutic agents can be present in the particles and / or compositions of the present application. In some embodiments, two or more different therapeutic agents are present in the same particle or in different, separate particles.

[0070] The therapeutic agent can be present in the granules of the present application in an amount of about 50%, 55%, 60%, 65%, or 70% to about 75%, 80%, 85%, 90%, 95%, or 99% by weight of the granule. In some embodiments, the granules comprise the therapeutic agent in an amount of about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% by weight of the granule. In some embodiments, the therapeutic agent is present in the granules in an amount of about 75% or 80% to about 85%, 90%, or 95% by weight of the granule, for example, in an amount of about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% by weight of the granule. In the granules of the present application, the hyaluronidase and the therapeutic agent can not be covalently bound and / or the excipient (e.g., stabilizer) can not be covalently bound to the therapeutic agent.

[0071] In some embodiments, the granules of the present application can be free of the therapeutic agent.

[0072] A stabilizer can be present in the granules of the present application and / or the compositions of the present application. Exemplary stabilizers include, but are not limited to, sugars, amino acids, polymers (e.g., hyaluronic acid, gelatin, and / or gelatin), antioxidants, surfactants, and / or proteins, such as globular proteins (e.g., albumin) and / or disordered proteins (e.g., intrinsically disordered proteins). In some embodiments, the stabilizer is albumin (e.g., serum albumin and / or recombinant albumin, such as that commercially available from Albumedix Ltd. of Nottingham, United Kingdom). In some embodiments, the stabilizer is hyaluronic acid. In some embodiments, the granules of the present application can comprise a sugar, such as, but not limited to, sucrose, glucose, trehalose, mannitol, sorbitol, dextrose, maltose, and / or lactose. In some embodiments, the granules of the present application can comprise an amino acid, such as, but not limited to, histidine, methionine, arginine, lysine, aspartic acid, glutamic acid, proline, glycine, and / or leucine. In some embodiments, the granules of the present application can comprise albumin, such as, but not limited to, serum albumin. One or more (e.g., 1, 2, 3, 4, or 5) different stabilizers can be present in the granules and / or compositions of the present application. In the granules of the present application, the hyaluronidase and the stabilizer can not be covalently bound and / or the stabilizer can not be covalently bound to the therapeutic agent.

[0073] The stabilizing agent can be present in the granules of the present application in an amount of about 0.1%, 0.5%, 1%, 5%, 10%, 15%, or 20% to about 25%, 30%, 35%, 40%, 45%, or 50% by weight of the granule. In some embodiments, the stabilizing agent can be present in the granules of the present application in an amount of about 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by weight of the granule. In some embodiments, the stabilizing agent (e.g., an amino acid) can be present in the granules of the present application in an amount of about 0.1%, 0.5%, 1%, 5%, or 10% to about 15%, 20%, 25%, or 30% by weight of the granule.

[0074] According to some embodiments of the present application, compositions comprising the granules of the present application are provided. The compositions can comprise a plurality of the granules of the present application. In some embodiments, the composition is a suspension, and one or more granules, which can be the same and / or different from each other, are suspended in a solvent (e.g., vehicle) present in the composition. In some embodiments, the composition is in the form of a powder and / or a solid.

[0075] In some embodiments, the composition (e.g., suspension) of the present application comprises a plurality of granules, and each of the plurality of granules comprises a hyaluronidase and a therapeutic agent. In some embodiments, the composition of the present application comprises a non-aqueous solvent (e.g., a non-aqueous vehicle) and a plurality of granules, wherein each of the plurality of granules comprises a hyaluronidase and a therapeutic agent. In some embodiments, the composition of the present application comprises an aqueous solvent (e.g., an aqueous vehicle) and a plurality of granules, wherein each of the plurality of granules comprises a hyaluronidase and a therapeutic agent. In some embodiments, the composition of the present application comprises a plurality of granules, wherein each of the plurality of granules comprises a hyaluronidase and a therapeutic agent, and wherein the composition is in the form of a powder.

[0076] In some embodiments, a composition of the present application (e.g., a suspension) comprises a plurality of particles, and a first portion of the plurality of particles comprises particles each comprising a hyaluronidase and no different therapeutic agent, and a second portion of the plurality of particles comprises particles each comprising a therapeutic agent different from the hyaluronidase and no hyaluronidase. In some embodiments, a composition of the present application comprises a non-aqueous solvent (e.g., a non-aqueous vehicle), a first plurality of particles, wherein each particle of the first plurality of particles comprises a hyaluronidase and no different therapeutic agent, and a second plurality of particles, wherein each particle of the second plurality of particles comprises a therapeutic agent different from the hyaluronidase and no hyaluronidase. In some embodiments, a composition of the present application comprises an aqueous solvent (e.g., an aqueous vehicle), a first plurality of particles, wherein each particle of the first plurality of particles comprises a hyaluronidase and no different therapeutic agent, and a second plurality of particles, wherein each particle of the second plurality of particles comprises a therapeutic agent different from the hyaluronidase and no hyaluronidase. In some embodiments, a composition of the present application comprises a first plurality of particles, wherein each particle of the first plurality of particles comprises a hyaluronidase and no different therapeutic agent, and a second plurality of particles, wherein each particle of the second plurality of particles comprises a therapeutic agent different from the hyaluronidase and no hyaluronidase, wherein the composition is in the form of a powder.

[0077] In some embodiments, a composition of the present application comprises a plurality of particles suspended in the composition (i.e., not dissolved in the composition) and one or more components (e.g., therapeutic agents) dissolved in a vehicle (e.g., solvent) present in the composition. In some embodiments, the one or more components (e.g., therapeutic agents) are dissolved in a non-aqueous vehicle present in the composition. In some embodiments, the one or more components (e.g., therapeutic agents) are dissolved in an aqueous vehicle present in the composition. The one or more components can be different from the hyaluronidase. In some embodiments, the hyaluronidase is not dissolved in the composition of the present application and / or the composition of the present application has no dissolved hyaluronidase. In some embodiments, a composition of the present application comprises a non-aqueous solvent; a plurality of particles of the present application, wherein each particle comprises a hyaluronidase and is suspended in the non-aqueous solvent; and a therapeutic agent that is not a hyaluronidase and is dissolved in the non-aqueous solvent.

[0078] The particles of the present application can be present as solids in the compositions of the present application. In some embodiments, the compositions can comprise the particles of the present application in an amount of from about 1, 5, 10, 25, 50, 75, 100, 150, or 200 mg particles (e.g., solids) per mL of the composition to about 300, 350, 400, 450, 500, 550, 600, 650, or 700 mg particles (e.g., solids) per mL of the composition. In some embodiments, the compositions are non-aqueous and / or the compositions comprise a non-aqueous vehicle. As used herein with respect to a composition or vehicle, "non-aqueous" refers to a composition or vehicle that is prepared from, prepared using, or prepared by means of a liquid other than water, and has the properties and / or characteristics of a liquid other than water. In some embodiments, the non-aqueous composition or vehicle comprises one or more (e.g., 1, 2, 3, 4, 5, or more) organic liquids that are insoluble in and / or immiscible or miscible with water. In some embodiments, the compositions of the present application comprise a non-aqueous organic solvent. In some embodiments, the compositions and / or vehicles of the present application comprise water. In some embodiments, the compositions and / or vehicles of the present application are anhydrous. As used herein, "anhydrous" means that water is not added directly to the composition or vehicle when it is prepared. However, one of skill in the art will recognize that at any point during the preparation, storage, and / or use of the composition or vehicle, water can be physically and / or chemically absorbed by one or more components of the composition or vehicle (i.e., water is added indirectly to the composition). In some embodiments, the term "anhydrous" means that the composition has a water content of less than 5% or any range and / or individual value therein by weight of the composition. The compositions of the present application can have a water content of less than 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, or 0.5% or any range therein by weight of the composition. Water content can be measured by methods known to one of skill in the art, for example, but not limited to, Karl Fischer titration.

[0079] The compositions of the present application can comprise a solvent. One or more (e.g., 1, 2, 3, 4, 5, or more) different solvents can be present in the compositions of the present application. The particles of the present application can be insoluble in the solvent present in the compositions of the present application, optionally wherein the particles are insoluble in the solvent after storage in the composition at a temperature of about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 °C to about 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 °C for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months or more. In some embodiments, the particles of the present application can be insoluble in the compositions of the present application, optionally wherein the particles are insoluble in the composition after storage at a temperature of about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 °C to about 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 °C for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months or more. In some embodiments, the hyaluronidase and / or the therapeutic agent are insoluble in the solvent and / or the composition of the present application. In some embodiments, the hyaluronidase is insoluble in the solvent and / or the composition of the present application, while the therapeutic agent is soluble in the solvent and / or the composition of the present application. Thus, the particles and / or the hyaluronidase of the present application can remain solid in the composition of the present application. Exemplary solvents that can be present in the compositions of the present application include, but are not limited to, alcohols, e.g., alcohols having 1-20 carbons (i.e., C1-C20 alcohols) or C4-C20 alcohols (e.g., propanol, pentanol, hexanol, etc.); alkanes, e.g., alkanes having 1-20 carbon atoms (i.e., C1-C20 alkanes) or C4-C20 alkanes (e.g., propane, pentane, hexane, etc.); aldehydes (e.g., C5-C14 aldehydes); acetates; esters (e.g., benzyl benzoate, fatty acid esters such as ethyl oleate, triacylglycerides such as 812 (saturated coconut / palm kernel oil-derived caprylic and capric fatty acids triacylglycerides with plant-derived glycerol) and / or 840 (esters of saturated plant-derived caprylic and capric fatty acids with propylene glycol, which can be referred to as propylene glycol dicaprylocaprate)); ethers; carboxylic acids; (poly)heteroatom cyclic, acyclic, straight-chain, or branched-chain molecules; oils (e.g., sesame oil, castor oil, soybean oil, and / or cottonseed oil); lactate esters, such as butyl lactate and / or ethyl lactate; and / or fluorinated compounds, such as perfluorocarbons (e.g., perfluorodecalin, perfluorooctane, and / or perfluooctylbromide) and / or semi-fluorinated alkanes (e.g., perfluorobutylbutane, perfluorobutylpentane, perfluorobutylhexane, perfluorobutyloctane, perfluorohexylhexane, perfluorohexyloctane, and / or perfluorohexyldodecane)). In some embodiments, the solvent can comprise carbon, nitrogen, and / or sulfur atoms. The compositions of the present application can comprise solvents that are esters, such as alkyl esters or aryl esters (e.g., benzyl benzoate). In some embodiments, the compositions of the present application comprise C1-C20 alcohols, ester-containing compounds (e.g., C1-C20 ester-containing compounds, such as, for example, ethyl acetate, butyl acetate, triacetin, isobutyl acetate, isopropyl acetate, isopropyl myristate, methyl acetate, propyl acetate, and / or butyl lactate), acetic acid, acetone, anisole, t-butyl methyl ether, isopropyl benzene, dimethyl sulfoxide, diethyl ether, ethyl formate, formic acid, alkanes (e.g., heptane, pentane, etc.), and / or methyl ethyl ketone. In some embodiments, the compositions of the present application comprise benzyl benzoate, ethyl oleate, triacylglyceride, ethyl lactate, and / or sesame oil. In some embodiments, the solvent is an aqueous solvent (e.g., water, saline, etc.).

[0080] The compositions of the present application can have a viscosity of greater than 1 cP when measured at 25 degrees Celsius. In some embodiments, the compositions of the present application can be a suspension comprising a plurality of particles suspended in one or more (e.g., 1, 2, 3, 4, 5, or more) non-aqueous phases. In some embodiments, the compositions of the present application can have a viscosity of greater than 1 cP when measured at a temperature of about 20 degrees Celsius and about 25 degrees Celsius, and a shear rate of about 1,000 s -1 to about 3,000 s -1The compositions of the present invention may have a viscosity of about 20, 30, 40, 50, 60, or 70 centipoise (cP) to about 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 cP when measured at a shear rate of 180°C.

[0081] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5 or more) different therapeutic agents may be present in the compositions of the invention. In some embodiments, the compositions of the invention comprise a first particle and a second particle, and the first particle comprises a first therapeutic agent and the second particle comprises a second therapeutic agent different from the first therapeutic agent, optionally wherein a hyaluronidase may be present in the first particle and / or the second particle. In some embodiments, the compositions of the invention comprise particles comprising a first therapeutic agent and / or a hyaluronidase suspended in the composition and a second therapeutic agent dissolved in a solvent present in the composition, wherein the first therapeutic agent (if present) is different from the second therapeutic agent.

[0082] One or more (e.g., 1, 2, 3, 4, 5 or more) different excipients can be present in the particles and / or compositions of the present invention. Exemplary excipients include, but are not limited to, stabilizers, surfactants (e.g., detergents), tonicity agents, sugars, salts, antimicrobials, antioxidants, reducing agents, and the like. In some embodiments, the excipient is present in the compositions of the present invention at a concentration of from about 0.01%, 0.05%, 0.1%, or 0.5% to about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% by weight of the composition. Exemplary surfactants include, but are not limited to, polysorbates (e.g., polysorbate-20 and / or polysorbate-80), sorbitan esters (Spans; e.g., sorbitan monostearate, sorbitan tristearate, sorbitan monolaurate, sorbitan trioleate, sorbitan monopalmitate, sorbitan monooleate, and / or sorbitan sesquioleate), pegylated fatty esters and ethers (e.g., Laureth-4), sucrose esters, block copolymers (e.g., poloxamers such as poloxamer 188), and / or ethoxylated triglycerides (e.g., ethoxylated castor oil).

[0083] Exemplary antimicrobial agents include, but are not limited to, benzoic acid and / or benzyl alcohol. Exemplary antioxidants include, but are not limited to, butylated hydroxyanisole, butylated hydroxytoluene, propyl gallate, t-butylhydroquinone, and / or vitamin E (e.g., a-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, a-tocotrienol, β-tocotrienol, γ-tocotrienol, and / or δ-tocotrienol). Exemplary reducing agents include, but are not limited to, glutathione and / or dithiothreitol (DTT).

[0084] The particles of the present application can be prepared by removing (e.g., dissolving and / or extracting) water from a hyaluronidase present in a composition (e.g., an aqueous composition) optionally comprising a therapeutic agent and / or an excipient to provide a solid particle. In some embodiments, the particles of the present application are prepared by contacting an aqueous phase and a solvent phase to dehydrate a hyaluronidase present in one phase and form a particle comprising the hyaluronidase. The methods of the present application can provide a solid particle comprising a hyaluronidase, an optional therapeutic agent, and an optional excipient, wherein the solid particle has a water content of less than about 15% by weight of the particle and / or a water activity of less than about 0.9. In some embodiments, the methods of the present application comprise removing water from a hyaluronidase present in a composition and providing a solidified hyaluronidase particle (i.e., a hyaluronidase in a solid form). Upon formation of the particles of the present application, the particles can be separated from any liquid and / or provided in a powder form. In some embodiments, the particles of the present application can be suspended in a solvent (e.g., a non-aqueous solvent).

[0085] In some embodiments, the particles of the present application can be prepared according to a micro- vitrification method. In some embodiments, water can be removed from a composition comprising a hyaluronidase using an aqueous phase and a non-aqueous phase to provide a solid comprising a hyaluronidase. In some embodiments, the compositions and / or methods (e.g., micro-vitrification methods) used herein do not include (i.e., are free of) a phase separation agent. In some embodiments, the particles of the present application are particles comprising micro-vitrified hyaluronidase. In some embodiments, the particles comprising a hyaluronidase can be dissolved back into the original composition (e.g., an aqueous solution), a bodily fluid, and / or an aqueous composition (e.g., an aqueous buffer) in which it was present. In some embodiments, the methods of forming the particles of the present application can include devices, steps, and / or compositions as described in U.S. Patent No. 8,013,022 and / or U.S. Patent Application Publication No. 2022 / 0119760, which are incorporated herein by reference in their entirety.

[0086] In some embodiments, a composition comprising a hyaluronidase (e.g., an aqueous composition) is contacted with a dehydrating composition to make the particles of the present application. As used herein, “contact,” “contacting,” “in contact,” and grammatical variations thereof refer to the bringing together of two or more materials (e.g., compositions, compounds, solvents, etc.) to form a mixture. The two or more materials can be brought into contact by pouring, spraying, mixing, flowing, injecting (e.g., microinjection), etc., the two materials or portions thereof together. For example, contacting can include adding a solvent to a composition comprising a hyaluronidase or adding a composition comprising a hyaluronidase to a solvent. Contacting can be performed in a device such as, for example, a mixer, a homogenizer, and / or a microfluidic device.

[0087] In some embodiments, contacting includes bringing two or more materials (e.g., compositions, compounds, solvents, etc.) close enough together such that a desired reaction can occur under suitable conditions (e.g., water can be removed from a component present in one of the materials). In some embodiments, the contacting step forms a mixture and the mixture can be one phase or two or more (e.g., 2, 3, or more) phases. In some embodiments, the mixture is a multi-phase composition because the composition has two or more (e.g., 2, 3, 4, or more) phases. In some embodiments, the contacting step forms a two-phase composition and / or an emulsion. In some embodiments, the contacting step forms a suspension (e.g., one or more liquid phases comprising solid particles). In some embodiments, an emulsion is formed and the emulsion can be a water-in-oil emulsion, optionally comprising droplets having a diameter and / or minimum dimension of less than about 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 25, 10, 5, or 1 pm. In some embodiments, the mixture is a water-in-oil emulsion comprising droplets having a diameter and / or minimum dimension in the range of about 5, 10, 25, 50, 100, 200, 300, 400, or 500 pm to about 600, 700, 800, 900, or 1000 pm. In some embodiments, the mixture is a water-in-oil emulsion comprising droplets having a diameter and / or minimum dimension in the range of about 0.1, 0.5, 1, 2, 3, 4, or 5 pm to about 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 pm. In some embodiments, the contacting step forms a mixture (e.g., an emulsion) and, as a result of the contacting step and / or forming the mixture, a component (e.g., a hyaluronidase) present in one of the materials is at least partially dehydrated.

[0088] The particles of the present application can be formed by contacting a composition comprising a hyaluronidase with a dehydrating composition that at least partially dehydrates a component present in the composition (e.g., a hyaluronidase, a therapeutic agent, and / or an excipient present in the composition). Upon contact of the dehydrating composition with the composition comprising a hyaluronidase, the dehydrating composition can be present in a concentration and / or volume sufficient to at least partially dehydrate a component of the composition (e.g., a hyaluronidase). In some embodiments, the dehydrating composition comprises a solvent (e.g., an organic solvent) that removes water from a component present in the composition (e.g., a hyaluronidase and / or a therapeutic agent). Exemplary organic solvents include, but are not limited to, alcohols such as, for example, alcohols having 1-20 carbons (i.e., C1-C20 alcohols) or C4-C20 alcohols, ester-containing compounds (e.g., C1-C20 ester-containing compounds such as, for example, ethyl acetate, butyl acetate, triacetin, isobutyl acetate, isopropyl acetate, methyl acetate, propyl acetate, and / or butyl lactate), acetic acid, acetone, anisole, t-butyl methyl ether, cumene, dimethyl sulfoxide, diethyl ether, ethyl formate, formic acid, alkanes (e.g., heptane, pentane, etc.), and / or methyl ethyl ketone.

[0089] In some embodiments, the dehydrating composition comprises a Ci, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, and / or C20 alcohol and / or a Ci, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, and / or C20 ester containing compound. In some embodiments, the alcohol is selected from the group consisting of methanol; ethanol; propanol (e.g., 1-propanol; 2-propanol); butanol (e.g., 1-butanol; 2-butanol); 2-methyl-l-propanol (proponal); 2-methyl-2-propanol; t-butanol; pentanol (e.g., 1-pentanol, 3-methyl-l-butanol, 2,2-dimethyl-l-propanol, cyclopentanol); hexanol (e.g., 1-hexanol); cyclohexanol; heptanol (e.g., 1-heptanol); octanol (e.g., 1-octanol); nonanol (e.g., 1-nonanol); decanol (e.g., 1-decanol); 2-propen-l-ol; benzyl alcohol; phenylmethanol; benzhydrol; undecanol; dodecanol; propyldecanol; butadecanol; pentadecanol; hexadecanol (e.g., 1-hexadecanol); and / or trityl alcohol. In some embodiments, the dehydrating composition does not comprise a C10 alcohol (e.g., 1-decanol). In some embodiments, the ester containing compound can be formed from an acid and a Ci-C2o, Ci-Cio, Ci-Cs, Ci-C6, or Ci-C4alcohol. In some embodiments, the dehydrating composition comprises an isomer of a straight chain alcohol (e.g., 2-octanol, 3-pentanol, 4-decanol), a derivative of a straight chain alcohol or isomer thereof (e.g., octyldodecanol, neopentyl alcohol), a di-, tri-, or tetra-hydroxylated material (e.g., 1,4-butanediol, glycerol), an unsaturated alcohol (e.g., a cyclic alcohol, an enol, or an alkynyl alcohol such as, for example, cyclohexanol, geraniol, oleyl alcohol), and / or an alcohol that incorporates internal and / or external heteroatoms (e.g., polyethylene glycol, polypropylene glycol, lactate esters, etc.).

[0090] One or more (e.g., 1, 2, 3, 4, or more) solvents can be present in the dehydrating composition of the present application. For example, in some embodiments, at least two alcohols (e.g., 1-pentanol and 1-hexadecanol), at least two ester-containing compounds (e.g., ethyl acetate and triacetin), or at least one alcohol (e.g., 1-pentanol) and one ester-containing compound (e.g., ethyl acetate) are present in the dehydrating composition. When two or more solvents are present in the dehydrating composition, they can be present in any suitable ratio. In some embodiments, the dehydrating composition comprises two solvents that are present in the composition in a ratio of about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20 (by volume or weight).

[0091] The solvents (e.g., organic solvents) present in the dehydrating composition of the present application can have a solubility in water of about 0.05%, 1%, 2%, or 5% to about 10%, 12%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% w / w. In some embodiments, the organic solvents have a solubility in water of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% w / w. The solvents (e.g., organic solvents) can have an interfacial tension with water and / or aqueous compositions of less than about 55 mN / m. In some embodiments, the solvents present in the dehydrating composition of the present application have an interfacial tension with water and / or aqueous compositions of less than about 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mN / m. In some embodiments, the solvents present in the dehydrating composition of the present application have an interfacial tension with water and / or aqueous compositions in the range of about 1, 2, 3, 4, or 5 to about 6, 7, 8, 9, or 10.

[0092] In some embodiments, the dehydrating composition of the present application consists of one or more (e.g., 1, 2, 3, 4, 5, or more) different organic solvents (e.g., alcohols). In some embodiments, the dehydrating composition consists of two or more C1-C20 alcohols. In some embodiments, the dehydrating composition of the present application comprises an organic solvent in an amount of about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% by weight of the dehydrating composition.

[0093] The dehydrated composition can comprise one or more (e.g., 1, 2, 3, 4, 5, or more) additives (e.g., stabilizers, surfactants, tonicity agents, salts, sugars, antimicrobial agents, antioxidants, etc.). Exemplary additives (e.g., surfactants, antimicrobial agents, antioxidants, etc.) include, but are not limited to, those described above. In some embodiments, the additive is present in the dehydrated composition at a concentration of about 0.01%, 0.1%, or 0.5% to about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% by weight of the dehydrated composition. In some embodiments, the dehydrated composition comprises a surfactant and / or a sugar. In some embodiments, the surfactant can be present in the dehydrated composition in an amount of about 30% or less by weight of the dehydrated composition, such as, for example, about 25%, 20%, 15%, 10%, 5% or less by weight of the dehydrated composition. In some embodiments, the antimicrobial agent can be present in the dehydrated composition in an amount of about 1% or less by weight of the dehydrated composition, such as, for example, about 0.5%, 0.1% or less by weight of the dehydrated composition. In some embodiments, the antioxidant can be present in the dehydrated composition in an amount of about 2.5% or less by weight of the dehydrated composition, such as, for example, about 2%, 1.5%, 1%, 0.5%, 0.1% or less by weight of the dehydrated composition.

[0094] In some embodiments, the dehydrating composition comprises at least one alcohol selected from benzyl alcohol, 1-propanol, 1-butanol, t-butyl, sec-butyl, 1-pentanol, 1-hexanol, 1-heptanol, 1-octanol, 1-nonanol, 1-decanol, 1-undecanol, 1-dodecanol, propyl decanol, tetradecanol, pentadecanol, and hexadecanol and / or at least one solvent selected from water, triacetin, benzyl alcohol, acetic acid, acetone, anisole, 2-butanol, butyl acetate, t-butyl methyl ether, cumene, dimethyl sulfoxide, ethanol, ethyl acetate, diethyl ether, ethyl formate, formic acid, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, 3-methyl-1-butanol, methyl ethyl ketone, 2-methyl-1-propanol, pentane, 2-propanol, propyl acetate, and t-butanol. Further exemplary solvents that can be present in the dehydrating composition include, but are not limited to, alkanes such as, for example, alkanes having 1-20 carbon atoms (i.e., C1-C20 alkanes) or C4-C20 alkanes (e.g., propane, pentane, hexane, etc.); alcohols (e.g., C5-C14 alcohols); acetates; esters; ethers; carboxylic acids; (poly)heteroatomic cyclic, acyclic, linear, or branched molecules; and / or lactates. In some embodiments, the dehydrating composition includes a solvent comprising carbon, nitrogen, and / or sulfur atoms.

[0095] The methods of the present application can comprise contacting the composition comprising hyaluronidase with one or more (e.g., 1, 2, 3, 4, or more) dehydrating compositions. Thus, the methods can comprise contacting the composition comprising hyaluronidase with one or more (e.g., 1, 2, 3, 4, or more) solvents such as, for example, a first organic solvent, a second organic solvent, etc. When two or more dehydrating compositions are used, the solvents present in the composition can be the same or different. In some embodiments, the solvents present in a dehydrating composition can be more volatile than the solvents in the immediately preceding dehydrating composition and / or can be volatile organic compounds (e.g., having a boiling point of about 0, 50, or 100 °C to about 150, 200, or 260 °C). In some embodiments, the dehydrating composition and the at least partially dehydrated component can be contacted one or more times (e.g., 1, 2, 3, 4, 5, or more times).

[0096] The composition comprising hyaluronidase can be contacted with one or more dehydrating compositions to produce the particles of the present application using a batch or inline process. In some embodiments, the methods comprise contacting the composition comprising hyaluronidase with a first dehydrating composition to form a mixture, and then contacting the mixture with a second dehydrating composition to form the particles of the present application. In some embodiments, the methods comprise contacting the composition comprising hyaluronidase with a first dehydrating composition to form a mixture, separating at least partially dehydrated hyaluronidase from the mixture, and contacting the at least partially dehydrated hyaluronidase with a second dehydrating composition, thereby forming the particles of the present application.

[0097] The solvent present in the dehydrating composition can form an interface with the composition comprising the hyaluronidase. In some embodiments, the solvent present in the dehydrating composition is immiscible with water. In some embodiments, the solvent present in the dehydrating composition first used to dehydrate the components (i.e., the first dehydrating composition or the first organic solvent) is immiscible with water and / or forms an interface with water, and the solvent is at least partially water-soluble. In some embodiments, the solvent present in the dehydrating composition used after the first dehydrating composition (e.g., the second or third dehydrating composition or the second or third organic solvent) is immiscible with water.

[0098] Contacting and / or forming a mixture of the dehydrated composition with the composition comprising a hyaluronidase can include reducing the water content of the components (e.g., hyaluronidase and / or therapeutic agent) present in the mixture by at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 111%, 1 3%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, after the contacting step of the present invention, the moisture content of the component is reduced by about 5%, 10%, 15%, 20%, 25%, 30%, or 35% to about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0099] The mixture comprising the dehydrated composition and component (e.g., hyaluronidase and / or therapeutic agent) can have a water activity of less than about 0.99, 0.98, 0.95, 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, or 0.1. In some embodiments, the mixture has a water activity in a range of about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 to about 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, or 0.95. In some embodiments, the mixture has a fractional water saturation of less than about 0.95, 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, or 0.1. In some embodiments, the mixture has a fractional water saturation in a range of about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 to about 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, or 0.95. For example, in some embodiments, a mixture comprising a protein can have a fractional water saturation of about 0.95 or less. In some embodiments, a mixture comprising a salt can have a fractional water saturation of about 0.5 or less.

[0100] The at least partially dehydrated component (e.g., hyaluronidase and / or therapeutic agent) can have a water content of about 99%, 98%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or 0% after contact with the one or more dehydrated compositions. In some embodiments, the at least partially dehydrated component (e.g., hyaluronidase) has a water content ranging from about 0%, 0.5%, 1%, 5%, 10%, 15%, 20%, or 25% to about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% after contact with the one or more dehydrated compositions. In some embodiments, the at least partially dehydrated component (e.g., hyaluronidase) has a water content ranging from about 0%, 0.5%, 1%, 2%, 3%, or 4% to about 5%, 6%, 7%, 8%, 9%, or 10% after contact with the one or more dehydrated compositions. In some embodiments, the at least partially dehydrated component (e.g., hyaluronidase) has a water activity of about 0.986, 0.98, 0.95, 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, 0.05, or 0 after contact with the one or more dehydrated compositions. In some embodiments, the at least partially dehydrated component (e.g., hyaluronidase) has a water activity ranging from about 0, 0.05, 0.1, 0.15, 0.2, 0.25, or 0.3 to about 0.35, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 after contact with the one or more dehydrated compositions. In some embodiments, the at least partially dehydrated component (e.g., hyaluronidase) has a water activity of less than about 0.75 after contact with the one or more dehydrated compositions. In some embodiments, the at least partially dehydrated component (e.g., hyaluronidase) has a water activity of less than about 0.5 after contact with the one or more dehydrated compositions.

[0101] In some embodiments, the at least partially dehydrated component (e.g., hyaluronidase) has a moisture content of less than about 60% and / or in a range of about 1%, 5%, 10%, 15%, 20%, or 25% to about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% after contacting the original dehydrating composition and the composition comprising hyaluronidase. In some embodiments, the at least partially dehydrated component has a moisture content of less than about 45% and / or in a range of about 0.5%, 1%, 5%, 10%, 15%, or 20% to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% after contacting the at least partially dehydrated component with a subsequent (e.g., second) dehydrating composition, which can be the same or different from the original dehydrating composition. In some embodiments, the at least partially dehydrated component is a particle of the present application.

[0102] The contacting step can be performed at a temperature in a range of about -50, -40, -30, -20, -10, 0, 4, 10, 15, or 20 °C to about 25, 20, 35, 40, 45, or 50 °C. In some embodiments, the contacting step in the methods of the present application is performed at room temperature and / or at atmospheric pressure.

[0103] In some embodiments, a suspension can be formed upon contacting the dehydrating composition (e.g., organic solvent) with the composition comprising hyaluronidase and / or forming a mixture comprising the dehydrating composition and the composition comprising hyaluronidase. In some embodiments, the suspension comprises hyaluronidase in solid form suspended in a liquid (e.g., non-aqueous organic phase). Contacting the dehydrating composition and the composition comprising hyaluronidase to provide the mixture and forming the at least partially dehydrated component in the mixture can be performed substantially simultaneously. As used herein with respect to at least partially dehydrating a component, “substantially simultaneously” means dehydrating the component upon contacting the dehydrating composition or within less than about 1 minute from the initial contacting with the dehydrating composition. In some embodiments, forming the at least partially dehydrated component in the mixture is achieved within about 100 minutes or less (e.g., about 90, 60, 30, 15, 10, 5, or 1 minute or less) from the initial contacting of the dehydrating composition and the composition comprising hyaluronidase.

[0104] In some embodiments, the at least partially dehydrated component (e.g., hyaluronidase) having a water content of less than about 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% can be obtained in less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute or less than about 50, 40, 30, 20, 10, 5, 1, 0.5, or 0.1 seconds. In some embodiments, the at least partially dehydrated component (e.g., hyaluronidase) having a water content of less than about 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% can be obtained in about 0.01, 0.05, 0.1, or 0.5 seconds to about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds.

[0105] According to some embodiments, contacting the two different compositions (e.g., the dehydrating composition and the composition comprising hyaluronidase, or the solvent and the composition comprising the at least partially dehydrated component) includes mixing them together. Mixing can be achieved by any method known in the art, such as, for example, vortexing, stirring, static mixing, homogenization, extrusion, pumping, injection (e.g., microinjection), adding one composition to another, spraying one composition into another, and / or spraying the compositions together to form a mixture. Mixing can form an emulsion. In some embodiments, air can be incorporated into the mixture. One composition can be added to another using slow addition, rapid addition, or all at once, and mixing can be achieved using low shear and / or high shear (e.g., about 1-10,000,000 / s). When the two different compositions include an aqueous composition and a hydrophobic composition (e.g., a composition comprising an organic solvent), the aqueous composition can be added to the hydrophobic composition, or vice versa. In some embodiments, the two different compositions are contacted prior to mixing and / or in a mixer. Exemplary mixers include, but are not limited to, high pressure homogenizers, rotor-stator homogenizers, high speed blenders, in-line mixers (turbulent and laminar), static mixers, inline mixers, and / or microfluidic devices (e.g., microfluidic devices comprising a cross junction). In some embodiments, the mixer is inline, such as, for example, an inline homogenizer. In some embodiments, the mixer is a device (e.g., a microfluidic device), and the two different compositions can be added or injected into the device to thereby mix the two different compositions together and form a mixture. Forming the at least partially dehydrated component can include mixing (e.g., homogenizing) the two different compositions and / or the mixture.

[0106] In some embodiments, two different compositions forming a mixture and / or composition comprising at least partially dehydrated components can be mixed, e.g., in a batch process, at a ratio of the volume of the first composition to the volume of the second composition. For example, for a first composition and a second composition to be contacted in a batch process to form a mixture, the volume ratio can be the volume of the first composition to be mixed to the volume of the second composition to be mixed. In some embodiments, two different compositions forming a mixture and / or composition comprising at least partially dehydrated components can be mixed, e.g., in a series process, at a ratio based on the feed rate of the two different compositions. For example, for a first composition and a second composition to be contacted in a series process to form a mixture, the ratio can be the feed rate (e.g., flow rate) of the first composition during the contacting step to the feed rate (e.g., flow rate) of the second composition during the contacting step.

[0107] In some embodiments, two different compositions forming a mixture and / or composition comprising at least partially dehydrated components can be mixed (e.g., homogenized) at a feed rate (e.g., flow rate) ranging from about 1, 5, 10, 25, 50, 75, or 100 mL / minute to about 150, 250, 500, 1,000, 1,500, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, or 8,000 mL / minute or ranging from about 5, 10, 25, 50, 75, or 100 L / minute to about 150, 250, 500, 750, 1,000, 1,500, 2,000, 3,000, 4,000, or 5,000 L / minute. In some embodiments, two different compositions forming a mixture and / or composition comprising at least partially dehydrated components can be mixed (e.g., homogenized) at a feed rate ranging from about 1, 5, 10, 25, 50, 75, or 100 L / hour to about 150, 250, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, or 4,000 L / hour or ranging from about 10,000, 15,000, 20,000, 25,000, or 30,000 L / hour to about 50,000, 75,000, 100,000, 125,000, or 150,000 L / hour. The feed rate of each composition can be the same or different.

[0108] In some embodiments, two different compositions forming a mixture and / or composition comprising at least partially dehydrated components can be contacted and / or combined in a given feed volume ratio. The two different compositions can include an aqueous-containing composition providing an aqueous phase (e.g., a composition comprising a hyaluronidase and / or a composition comprising water) and a solvent-containing composition providing a solvent phase (e.g., a hydrophobic composition). In some embodiments, the two different compositions can be contacted and / or combined in a feed volume ratio of about 0.1 : 100, 0.2: 100, 0.3: 100, 0.4: 100, 0.5: 100, 0.6: 100, 0.7: 100, 0.8: 100, 0.9: 100, 1 : 100, 1.5: 100, 2: 100, 2.5: 100, 3: 100: 3.5: 100, 4: 100, 4.5: 100, 5: 100, 5.5: 100, 6: 100, 6.5: 100, 7: 100, 7.5: 100, 8: 100, 8.5: 100, 9: 100, 9.5: 100, 10: 100, 11 : 100, 12: 100, 13: 100: 14: 100, 15: 100, 16: 100, 17: 100, 18: 100, 19: 100, 20: 100, 21 : 100, 22: 100, 23: 100: 24: 100, or 25: 100 (aqueous-containing composition: solvent-containing composition). In some embodiments, the two different compositions forming a mixture and / or composition comprising at least partially dehydrated components can be contacted and / or combined in a feed volume ratio ranging from about 0.1 : 100, 0.3: 100, 0.5: 100, 0.7: 100, or 1 : 100 to about 2: 100, 4: 100, 6: 100, 8: 100, 10: 100, 12: 100, 14: 100, 16: 100, 18: 100, 20: 100, 22: 100, or 25: 100.

[0109] The at least partially dehydrated components and / or particles of the present application can be separated from the mixture and / or liquid phase using methods known to those of skill in the art. For example, the at least partially dehydrated components and / or particles of the present application can be separated from the liquid phase by filtration (e.g., pressure filtration, tangential flow filtration, spin filtration, centrifugal filtration, disc stack filtration, etc.), cyclonic separation, sedimentation (e.g., by acoustic resonance field sedimentation), evaporation, centrifugation, fluidized bed drying, evaporative drying, thermal drying, freeze drying, atmospheric freeze drying, spray drying, spray freeze drying, microwave / IR drying, and / or sieving. In some embodiments, the at least partially dehydrated components and / or particles of the present application can be separated from the liquid phase by a washing step and / or solvent exchange method, sedimentation, and / or filtration step, and / or further dried (e.g., to remove water and / or solvent) using air, vacuum, and / or freeze drying steps and / or any combination thereof.

[0110] In some embodiments, the methods of the present application comprise washing the at least partially dehydrated components and / or particles of the present application with a washing composition. The washing composition can comprise an organic solvent, such as, for example, an alcohol (e.g., a C1-C20 alcohol), an alkane (e.g., a C4-C20 alkane), an ester, and / or an ether. Such organic solvents include, but are not limited to, those described above and / or the organic solvents can have a solubility in water as described above. In some embodiments, the organic solvent in the washing composition can be miscible with water. The organic solvent in the washing composition can be the same or different than the organic solvent in the dehydrating composition. In some embodiments, the organic solvent present in the washing composition can be a Class II or Class III residual solvent as classified by the U.S. Food and Drug Administration.

[0111] In some embodiments, the organic solvent present in the washing composition can be more volatile than the organic solvent in the dehydrating composition. In some embodiments, the organic solvent in the washing composition is a volatile organic compound (e.g., having a boiling point of about 0, 50, or 100 °C to about 150, 200, or 260 °C). In some embodiments, the washing composition can be used to wash off and / or remove the dehydrating composition, the organic solvent, and / or impurities. In some embodiments, the organic solvent present in the washing composition has a solubility in the solvent present in the dehydrating composition.

[0112] According to some embodiments, the methods of the application are one-step methods in which a composition comprising a hyaluronidase is contacted with a dehydrating composition to provide the mixture and particles of the application. The contacting step in a one-step method can be repeated one or more times (e.g., 1, 2, 3, 4, 5, or more times) with the same or different dehydrating composition. A one-step method can further comprise contacting the at least partially dehydrated component or a composition comprising the component with a washing composition, which optionally comprises an organic solvent that is more volatile than the previous organic solvent(s) used in the method.

[0113] The methods of the application can contact a dehydrating composition or a washing composition with a composition comprising a hyaluronidase, an at least partially dehydrated component, or a composition comprising an at least partially dehydrated component for a period of time. The contacting time for each of these steps can be about 1, 5, 15, 30, 45, or 60 minutes, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or more. In some embodiments, the contacting time is about 1, 5, or 15 minutes to about 30, 45, or 60 minutes or about 1, 2, 3, 4, or 5 hours to about 6, 7, 8, 9, or 10 hours or more.

[0114] In some embodiments, a composition comprising an at least partially dehydrated component (e.g., a particle of the application) has a pH greater than 7, such as, for example, about 7.5, 8, 8.5, 9, 10, or more. In some embodiments, a composition comprising an at least partially dehydrated component has a pH less than 7, such as, for example, about 6.5, 6, 5.5, 5, 4.5, 4, or less. In some embodiments, a composition comprising an at least partially dehydrated component has a pH that is at least ± 0.5, 1, 1.5, or 2 pH units from the isoelectric point (pi) of the desired isolated component (e.g., a hyaluronidase). In some embodiments, the pH of a composition comprising an at least partially dehydrated component in a method of the application varies by less than about ± 2, 1.5, 1, or 0.5 pH units from the initial pH of the composition.

[0115] According to some embodiments of the application, a composition comprising a plurality of solid particles (e.g., microparticles, nanoparticles) as described herein is provided. In some embodiments, the composition is non-aqueous. The plurality of solid particles comprised in the composition can be amorphous and / or crystalline. In some embodiments, the plurality of solid particles comprises amorphous particles. In some embodiments, the plurality of solid particles can comprise crystalline particles, such as, for example, when a small molecule (e.g., a salt) that can crystallize is present. In some embodiments, the composition can be provided and / or obtained after the solid particles are separated from a liquid phase. The plurality of solid particles can be uniform in size, or can be polydisperse. In some embodiments, at least a portion of the plurality of solid particles have a size within about ±5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, or more of the average particle size. In some embodiments, the composition comprises discrete particles. In some embodiments, the composition comprises less than about 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% of an amount of aggregation of a biologic (e.g., hyaluronidase and / or protein) by weight of the biologic.

[0116] In some embodiments, the particles of the application have a size configured to and / or capable of passing through pores created in an acetyl hyaluronic acid network when a hyaluronidase (e.g., a hyaluronidase present in and / or from the particles of the application) breaks down the acetyl hyaluronic acid network, which can allow the particles of the application to move away from a site (e.g., an injection site) to which the particles are administered to a subject. In some embodiments, the particles of the application have a size of about 50 nm to about 500 nm or about 100 nm to about 300 nm or about 100 nm to about 200 nm or about 200 nm to about 400 nm. In some embodiments, the plurality of particles of the application have an average size of about 50 nm to about 500 nm or about 100 nm to about 300 nm or about 100 nm to about 200 nm. In some embodiments, the particles of the application move and / or disperse an increased distance from an administration site compared to one or more same compounds present in particles that are not administered in the form of the particles of the application. In some embodiments, the particles of the application do not disperse from an administration site (e.g., an injection site) and / or reside locally at the administration site. Upon dissolution of the administered particles of the application, components of the particles (e.g., hyaluronidase and / or a therapeutic agent) can disperse from the administration site. In some embodiments, the particles of the application have a size of about 300 nm or greater, and the particles do not disperse from an administration site (e.g., an injection site) and / or reside locally at the administration site.

[0117] In some embodiments, at least a portion of the plurality of solid particles in the composition of the application are solid particles of the application comprising solidified hyaluronidase. In some embodiments, at least a portion of the plurality of solid particles in the composition of the application are solid particles comprising solidified therapeutic agent, optionally having a moisture content of less than about 15% by weight of the particle and / or a water activity of less than about 0.9. In some embodiments, at least a portion of the plurality of solid particles in the composition of the application comprise both solidified hyaluronidase and solidified therapeutic agent, either contained in the same particle or different particles. In some embodiments, the composition comprising a plurality of solid particles comprises at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100% by weight of the composition of solid particles comprising solidified hyaluronidase. In some embodiments, the solid particles comprising solidified hyaluronidase can be present in the composition in an amount of about 1, 5, 10, 25, 50, 75, 100, 150, or 200 mg / mL to about 300, 400, 500, 600, or 700 mg / mL. In some embodiments, the composition comprising a plurality of solid particles comprises at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100% by weight of the composition of solid particles comprising solidified therapeutic agent. In some embodiments, the plurality of solid particles have a moisture content of less than about 15% by weight of the particle and / or a water activity of less than about 0.9. In some embodiments, the plurality of solid particles have a moisture content of less than about 10% by weight of the particle and / or a water activity of less than about 0.5.

[0118] According to embodiments of the application, there is provided a method of increasing solubility of a therapeutic agent in a liquid, the method comprising combining the liquid, the therapeutic agent, and a particle comprising hyaluronidase of the application. In some embodiments, the particle comprising hyaluronidase is dissolved in the liquid. In some embodiments, the liquid is an aqueous liquid (e.g., a bodily fluid and / or an aqueous buffer). The therapeutic agent can be present in the particle comprising hyaluronidase or can be separate from the particle comprising hyaluronidase. In some embodiments, the therapeutic agent can be in a different particle than the particle comprising hyaluronidase, and the particle comprising the therapeutic agent can have a water content of less than about 15% by weight of the particle and / or a water activity of less than about 0.9, and can be a solid particle. In some embodiments, the therapeutic agent can be separate from the particle comprising hyaluronidase, and can be present (e.g., dissolved and / or suspended) in a composition in which the particle is present. In some embodiments, the particle comprising hyaluronidase is present in a solvent that is different from the liquid. In some embodiments, the therapeutic agent is present in a non-aqueous composition, and the particle is present in a non-aqueous composition, optionally wherein the therapeutic agent and the particle are present in the same composition or in different compositions. In some embodiments, the therapeutic agent is present in an aqueous composition. In some embodiments, the therapeutic agent is present in an aqueous composition, and the particle is present in a non-aqueous composition.

[0119] In some embodiments, a method of increasing solubility of a therapeutic agent in a liquid (e.g., in vitro and / or in vivo) can comprise independently combining the therapeutic agent and the particle comprising hyaluronidase with the liquid. For example, the therapeutic agent and the particle comprising hyaluronidase can be combined (e.g., added to) the liquid sequentially. In some embodiments, the combining comprises simultaneously combining the particle comprising hyaluronidase and the therapeutic agent in the liquid, optionally wherein the hyaluronidase and the therapeutic agent are present in the same particle or in different particles. Prior to and / or upon combining the liquid with the particle comprising hyaluronidase, the liquid and / or a composition comprising the particle can be free of hyaluronidase in solution (e.g., hyaluronidase dissolved in water) and / or free hyaluronidase. Upon combining the liquid and the particle comprising hyaluronidase, the hyaluronidase and / or the particle can be dissolved in the liquid.

[0120] The methods of the present application can increase dissolution of a therapeutic agent in a liquid by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, or more over a period of time (e.g., about 1 minute to about 2 hours) as compared to dissolution of the therapeutic agent in the same liquid under the same conditions (e.g., temperature and / or pressure) without the particles of the present application. In some embodiments, at least about 90%, 95%, 99%, or 100% of the therapeutic agent dissolves within about 10, 20, 30, or 40 minutes to about 50, 60, 70, 80, 90, 100, 110, or 120 minutes after the initial combination of the particles comprising hyaluronidase, the therapeutic agent, and the liquid of the present application. In some embodiments, at least about 90%, 95%, 99%, or 100% of the therapeutic agent dissolves within about 10, 20, 30, or 40 minutes to about 50 or 60 minutes after the initial combination of the particles comprising hyaluronidase, the therapeutic agent, and the liquid of the present application.

[0121] According to some embodiments of the present application, a method of improving tolerability and / or increasing absorption of a therapeutic agent upon administration to a subject is provided, the method comprising administering to the subject a particle comprising hyaluronidase of the present application. In some embodiments, the method comprises subcutaneously, intramuscularly, and / or intradermally administering to a subject a therapeutic agent and / or a particle comprising hyaluronidase. In some embodiments, the therapeutic agent and / or the particle can be administered to the subject via subcutaneous administration, intramuscular administration, intradermal administration, and the like using methods and / or devices known in the art. Exemplary devices include, but are not limited to, syringes (e.g., primed syringes with staked needles or removable needles, dual chamber syringes, plastic syringes filled from a vial by the subject or a caregiver), cartridges, and / or vials. In some embodiments, the syringe or cartridge can be used with an auto-injector or pen device. Exemplary needle gauges for use with devices (e.g., syringes) include, but are not limited to, 27G, 27G thin wall, 27G ultra thin wall, 21G, 23G, 25G, 26G, 29G, and / or 30G. In some embodiments, the therapeutic agent and / or the particle of the present application is injected subcutaneously into the subject. In some embodiments, the method reduces irritation to the subject at the site of administration (e.g., the site of injection).

[0122] In some embodiments, the therapeutic agent and the particle comprising a hyaluronidase are administered to the subject separately, optionally simultaneously or at different times (e.g., sequentially). In some embodiments, the therapeutic agent is administered to the subject first, and then the particle comprising a hyaluronidase is administered to the subject. In some embodiments, the particle comprising a hyaluronidase is administered to the subject first, and then the therapeutic agent is administered to the subject. In some embodiments, when the therapeutic agent and the particle comprising a hyaluronidase are administered separately, the time period between the two administrations can be less than about 24 hours, e.g., less than about 20, 15, 12, 8, 6, 4, 2, or 1 hour, or less than about 45, 30, 15, 10, 5, or 2 minutes. In some embodiments, the therapeutic agent and the particle comprising a hyaluronidase are administered to the subject simultaneously. In some embodiments, the therapeutic agent and the particle comprising a hyaluronidase are present in the same composition administered to the subject. In some embodiments, the therapeutic agent is present in a non-aqueous composition, and the particle is present in a non-aqueous composition, optionally wherein the therapeutic agent and the particle are present in the same composition or in different compositions. In some embodiments, the therapeutic agent and the hyaluronidase are present in the same particle administered to the subject or in separate particles administered to the subject. In some embodiments, the therapeutic agent and the particle comprising a hyaluronidase are both administered subcutaneously to the subject. In some embodiments, the therapeutic agent and the particle comprising a hyaluronidase are both administered intradermally to the subject. In some embodiments, the therapeutic agent is administered subcutaneously to the subject, and the particle comprising a hyaluronidase is administered intradermally to the subject. In some embodiments, the therapeutic agent is administered intradermally to the subject, and the particle comprising a hyaluronidase is administered subcutaneously to the subject. In some embodiments, the therapeutic agent and / or the particle comprising a hyaluronidase are administered intramuscularly to the subject.

[0123] The particles and / or therapeutic agents of the present application can be administered to a subject in a volume of about 0.1, 0.2, 0.3, 0.4, or 0.5 mL to about 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mL. In some embodiments, a composition comprising the particles and / or therapeutic agents of the present application can be administered to a subject, the composition having a volume of about 0.1, 0.2, 0.3, 0.4, or 0.5 mL to about 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mL. In some embodiments, the compositions of the present application administered to a subject have a volume of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mL. In some embodiments, the particles and therapeutic agents of the present application are administered to a subject in a total administration volume (i.e., the sum of the volume of the composition comprising the particles and the volume of the composition comprising the therapeutic agent) of less than about 10 mL, for example, less than about 9, 8, 7, 6, 5, 4, 3, 2, or 1 mL. In some embodiments, the particles and therapeutic agents of the present application are administered to a subject in a total volume of less than about 2 mL.

[0124] The methods of the present application can be free of the administration of hyaluronidase in solution (e.g., hyaluronidase dissolved in water) and / or free hyaluronidase. In some embodiments, the particles and / or hyaluronidase of the present application are in solid form at the time of administration to the subject (e.g., subcutaneous and / or intradermal administration). In some embodiments, the hyaluronidase included in the particles of the present application and / or the particles at the time of administration to the subject, e.g., upon contact with the bodily fluids of the subject, dissolve. The hyaluronidase can be administered to the subject in an amount of about 0.1%, 0.5%, or 1% to about 2%, 3%, 4%, or 5% by weight of the particle. In some embodiments, the methods of the present application can include administration of a composition to the subject that includes particles containing hyaluronidase, and the hyaluronidase can be administered to the subject and / or present in the composition in an amount of about 0.05%, 0.1%, 0.5%, or 1% to about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% by weight of the composition. In some embodiments, the hyaluronidase can be administered to the subject in an amount of about 100 activity units to about 50,000 activity units (e.g., about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,250, 3,500, 3,750, 4,000, 4,250, 4,500, 4,750, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, or 50,000 activity units). In some embodiments, the hyaluronidase activity in the compositions of the present application ranges from about 1, 2, 3, 4, 5 units / mg therapeutic agent to about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 units / mg therapeutic agent. In some embodiments, the hyaluronidase activity in the compositions of the present application ranges from about 10, 11, 12, 13, 14, or 15 units / mg therapeutic agent to about 16, 17, 18, 19, or 20 units / mg therapeutic agent. In some embodiments, the hyaluronidase is present in the compositions of the present application in an amount of about 1,000, 2,000, 3,000, or 4,000 units / mL of the composition to about 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, or 15,000 units / mL of the composition. In some embodiments, the hyaluronidase is present in the compositions of the present application in an amount of about 1,000, 1,500, or 2,000 units / mL of the composition to about 2,500, 3,000, 3,500, or 4,000 units / mL of the composition.In some embodiments, at least about 90%, 95%, 99%, or 100% of the therapeutic agent, hyaluronidase, and / or particle dissolves (e.g., in a bodily fluid) within about 1, 5, 10, 20, 30, or 40 minutes to about 50, 60, 70, 80, 90, 100, 110, or 120 minutes or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or more after administration to the subject. In some embodiments, at least about 90%, 95%, 99%, or 100% of the therapeutic agent, hyaluronidase, and / or particle dissolves within about 5, 10, 20, 30, or 40 minutes to about 50 or 60 minutes after administration to the subject. In some embodiments, at least about 90%, 95%, 99%, or 100% of the therapeutic agent, hyaluronidase, and / or particle has dissipated and / or dispersed from the site of administration within about 5, 10, 20, 30, or 40 minutes to about 50, 60, 70, 80, 90, 100, 110, or 120 minutes or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or more after administration to the subject.

[0125] In some embodiments, the methods increase the dispersion of the particles, therapeutic agent, and / or composition administered in the subject when the therapeutic agent and particles comprising hyaluronidase are administered to the subject. For example, in some embodiments, the methods increase the dispersion of the particles, therapeutic agent, and / or composition under the skin of the subject. This can be determined by methods known in the art, for example, by measuring the length of a bump, blister, and / or wheal under the skin after administration. In some embodiments, the therapeutic agent administered to the subject according to the methods of the application can have increased absorption, bioavailability, distribution, metabolism, and / or excretion in the subject compared to the absorption, bioavailability, distribution, metabolism, and / or excretion of the therapeutic agent in the subject after administration in the absence of the particles of the application.

[0126] In some embodiments, the methods of the application comprise administering to the subject a therapeutically effective amount of the therapeutic agent, hyaluronidase, particles, and / or composition of the application. As used herein, the term "therapeutically effective amount" refers to the amount of the therapeutic agent, hyaluronidase, particles, and / or composition of the application that elicits the therapeutically useful response in the subject. Those of skill in the art will appreciate that the therapeutic effect need not be complete or curative, as long as some benefit is provided to the subject.

[0127] As used herein, "treat," "treating," or "treatment" (and grammatical variations thereof) refer to any type of treatment that imparts a benefit to a subject, and can mean that the severity of the subject's condition (e.g., disease or disorder) is reduced, at least partially improved or ameliorated and / or that some alleviation, mitigation or decrease in at least one clinical symptom associated with the subject's condition is achieved and / or that there is a delay in the progression of the symptoms. In some embodiments, the severity of the symptoms associated with the subject's condition in the subject can be reduced as compared to the severity of the symptoms without the methods of the present application. In some embodiments, pain, swelling, and / or irritation of the injection site in the subject is reduced in the methods of the present application.

[0128] In some embodiments, the therapeutic agents, hyaluronidases, particles, and / or compositions of the present application can be administered in a therapeutically effective amount. As used herein, a "therapeutically effective" amount is an amount sufficient to treat (as defined herein) a subject. Those skilled in the art will appreciate that the therapeutic effect need not be complete or curative, as long as some benefit is provided to the subject. In some embodiments, a therapeutically effective amount can be achieved by administering the therapeutic agents, hyaluronidases, particles, compositions of the present application.

[0129] The terms "prevent," "preventing," and "prevention" (and grammatical variations thereof) refer to avoiding, reducing, and / or delaying the onset of symptoms associated with a condition (e.g., disease and / or disorder), and / or reducing the severity of the onset of symptoms associated with a condition as compared to what would occur in the absence of the methods of the present application. Prevention can be complete, e.g., complete absence of symptoms. Prevention can also be partial, such that the occurrence and / or severity of symptoms in the subject is less than what would occur in the absence of the methods of the present application. In some embodiments, the methods of the present application prevent or avoid pain, swelling, and / or irritation of the injection site in the subject.

[0130] In some embodiments, the therapeutic agents, hyaluronidases, particles, and / or compositions of the present application can be administered in a prophylactically effective amount. As used herein, a "prophylactically effective" amount is an amount sufficient to prevent (as defined herein) symptoms associated with a condition in a subject. Those skilled in the art will appreciate that the prophylactic level need not be complete, as long as some benefit is provided to the subject. In some embodiments, a prophylactically effective amount can be achieved by administering the therapeutic agents, hyaluronidases, particles, and / or compositions of the present application.

[0131] The present application has applications in both veterinary and medical applications. Subjects suitable for treatment with the methods of the present application include, but are not limited to, mammalian subjects. Mammals of the present application include, but are not limited to, canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g., rats and mice), lagomorphs, primates (e.g., simians and humans), non-human primates (e.g., monkeys, baboons, chimpanzees, gorillas), and the like, as well as mammals in utero. Any mammalian subject in need of treatment according to the present application is suitable. Human subjects of both sexes and at any stage of development (i.e., neonatal, infant, juvenile, adolescent, adult) can be treated according to the present application. In some embodiments of the present application, the subject is a mammal, and in certain embodiments, the subject is a human. Human subjects include both males and females of all ages, including fetal, neonatal, infant, juvenile, adolescent, adult, and geriatric subjects, as well as pregnant subjects. In particular embodiments of the present application, the subject is a human adolescent and / or adult.

[0132] The methods of the present application can also be performed on animal subjects, particularly mammalian subjects, such as mice, rats, dogs, cats, livestock, and horses, for veterinary purposes and / or for drug screening and drug development purposes.

[0133] In some embodiments, a subject "needs" or "has a need for" the methods of the present application, e.g., the subject has a finding generally associated with a condition, is suspected of having a condition, and / or the subject has a condition.

[0134] In some embodiments, the methods of the application can provide increased dispersion, dissolution, and / or dissipation of a therapeutic agent, a composition comprising a therapeutic agent, a vehicle, and / or an injected particle at a concentration of hyaluronidase that is reduced (lower) than the concentration of hyaluronidase that would be required for a method that is not in accordance with the application (e.g., a method that does not administer a particle of the application, e.g., a method that administers a hyaluronidase that is not in a particle of the application and / or a method that administers a hyaluronidase that is dissolved in an aqueous solution and / or a method that does not include and / or administer a hyaluronidase). For example, a method of the application that includes administering a particle of the application comprising a first concentration of hyaluronidase can allow a composition comprising a therapeutic agent and / or a therapeutic agent to be administered to a subject and to dissipate and / or dissolve within about 1 hour after administration of the therapeutic agent, whereas a method that is not in accordance with the application (e.g., a method that administers a hyaluronidase that is not in a particle of the application and / or a method that administers a hyaluronidase that is dissolved in an aqueous solution and / or a method that does not include and / or administer a hyaluronidase) must use a concentration of hyaluronidase that is higher than the first concentration of hyaluronidase in order to cause the same composition comprising a therapeutic agent and / or therapeutic agent to dissipate and / or dissolve at the same time. In some embodiments, a method of the application can use (e.g., administer to a subject) a reduced (e.g., at least about 5% or more reduced) concentration of hyaluronidase as compared to a method that is not in accordance with the application, optionally to obtain similar results (e.g., measurable values within ± 20%) as a method of the application. In some embodiments, a method of the application can use (e.g., administer to a subject) a reduced concentration of hyaluronidase as compared to a method that administers a free hyaluronidase and / or a method that administers a hyaluronidase that is present in an aqueous composition, optionally to obtain similar results (e.g., measurable values within ± 20%) as a method of the application.

[0135] In some embodiments, the methods of the application can provide an increased (e.g., increased by at least about 5%; e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) volume of a therapeutic agent and / or a composition comprising a therapeutic agent to be administered to a subject, optionally to obtain similar results (e.g., measurable values within ±20%) or improved results as the methods of the application, as compared to methods that do not follow the methods of the application (e.g., administering a hyaluronidase that is not in a particle of the application and / or administering a hyaluronidase that is dissolved in an aqueous solution and / or methods that do not include and / or administering a hyaluronidase). Similar results can be similar dissolution profiles, dispersion, bioavailability, swelling volume, side effects, pain, irritation at the injection site, etc. In some embodiments, the methods of the application can administer an increased (e.g., increased by at least about 5%; e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) volume of a therapeutic agent or a composition comprising the therapeutic agent, optionally to obtain similar results (e.g., measurable values within ±20%) or improved results as the methods of the application, as compared to methods that administer a free hyaluronidase and / or methods that administer a hyaluronidase that is present in an aqueous composition. In some embodiments, the methods of the application can administer an increased (e.g., increased by at least about 5%; e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) total administered volume to a subject, optionally to obtain similar results (e.g., measurable values within ±20%) or improved results as the methods of the application, as compared to methods that administer a free hyaluronidase and / or methods that administer a hyaluronidase that is present in an aqueous composition. In some embodiments, the methods of the application can administer an increased (e.g., increased by at least about 5%; e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) volume of a therapeutic agent or a composition comprising the therapeutic agent, as compared to methods that do not administer a hyaluronidase. In some embodiments, the methods of the application can administer an increased (e.g., increased by at least about 5%; e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) total administered volume to a subject, as compared to methods that do not administer a hyaluronidase.

[0136] In some embodiments, the methods of the application can provide an increased (e.g., increased by at least about 5%; e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) rate of dispersion, dissolution, and / or dissipation of a therapeutic agent, a composition comprising a therapeutic agent, a vehicle, and / or a particle injected compared to a method that does not comprise a method according to the application (e.g., administering a hyaluronidase that is not in a particle of the application and / or administering a hyaluronidase that is dissolved in an aqueous solution and / or a method that does not include and / or administering a hyaluronidase). In some embodiments, a composition comprising a particle of the application can provide an increased (e.g., increased by at least about 5%; e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) rate of dispersion, dissolution, and / or dissipation of a therapeutic agent, a composition comprising a therapeutic agent, a vehicle, and / or a particle injected compared to a composition that does not comprise a particle of the application (e.g., administering a hyaluronidase that is not in a particle of the application and / or administering a hyaluronidase that is dissolved in an aqueous solution).

[0137] In some embodiments, the methods of the application can administer (e.g., via injection into a subject) an increased (e.g., increased by at least about 5%; e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) volume of a total administration of a composition (e.g., a non-aqueous formulation) to a subject compared to a method that administers a free hyaluronidase and / or a method that administers a hyaluronidase dissolved in an aqueous solution. In some embodiments, a composition comprising a particle of the application can allow for an increased (e.g., increased by at least about 5%; e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) volume of a total administration of a composition (e.g., a non-aqueous formulation) to a subject compared to a composition that does not comprise a particle of the application.

[0138] In some embodiments, a composition of the application comprising hyaluronidase particles of the application can provide for an increased injection volume of the composition as compared to the injection volume of a non-aqueous composition without the particles of the application. In some embodiments, a composition of the application (e.g., a composition comprising particles of the application containing hyaluronidase) can provide for a lower injection force and / or a lower inline pressure during injection of the composition as compared to the injection force and / or inline pressure of a non-aqueous composition without the particles of the application. In some embodiments, a composition of the application comprising hyaluronidase particles of the application can provide for an increased dispersion and / or absorption of one or more components of the composition (e.g., an increased dispersion and / or absorption of solvent, therapeutic agent, and / or particles) as compared to the dispersion and / or absorption of one or more components in a non-aqueous composition without the particles of the application. In some embodiments, a composition of the application comprising hyaluronidase particles of the application can provide for an increased stability of one or more components in the composition as compared to the stability of one or more components in a non-aqueous composition without the particles of the application.

[0139] The application will now be discussed in the following non-limiting examples. Examples

[0140] Example 1 Particles containing hyaluronidase, sucrose, and bovine gamma globulin (BGG) were produced using a microfluidization method. Three batches were produced. The resulting particles contained about 2% hyaluronidase by weight of the particles. Particles were prepared in 17-mM Trizma buffer (SIGMA T1503-500G lot 068K5451) containing hyaluronidase type 5 (SIGMA H6254-500MG lot SLCK4168) and sucrose (SIGMA 1.00892.1003 lot K5289579203) with and without the addition of bovine gamma globulin (RMBIO BGG-BBZ-01K lot 20150424IG) using a small scale microfluidization method. Three stock solutions were used. The first contained no hyaluronidase, but contained 21 mg / g sucrose and 77 mg / g bovine gamma globulin. The second stock solution contained 21 mg / g sucrose, 71 mg / g bovine gamma globulin, and 10 mg / g hyaluronidase. The third stock solution contained 20 mg / g sucrose, no bovine gamma globulin, and 80 mg / g hyaluronidase. Particles were produced by mixing 10 to 12 microliters of the hyaluronidase stock solution with 1 milliliter of anhydrous n-octanol (f-value = 0.3). The particles were formed by shearing for 30 seconds using a homogenizer (IKA T-10) at setting "6". Once formed, a small sample of the suspension was taken for particle analysis. The suspension was centrifuged at 14,000 rcf for 10 minutes, and the octanol was carefully removed with a pipette. The particles were washed or decanted three times with n-pentanol by adding, mixing, centrifuging (using the same settings as described above), and then carefully removing the supernatant. The particles were dried under vacuum overnight, and then sealed and stored at 5°C. The particle morphology was spherical, and will be tested to determine enzyme activity.

[0141] Example 2 Dry, dense particles containing hyaluronidase, sucrose, and bovine gamma globulin (BGG) were produced using a microfluidization method. Three batches were produced. The resulting particles contained about 2% hyaluronidase by weight of the particles.

[0142] For the first batch, a stock solution of the protein was prepared at about 100 mg / ml total solids concentration. The octanol and aqueous stock solution were fed into a tandem homogenizer (IKA T-25) with a custom feed having a total flow rate speed of 176 ml / minute, of which 2.228 ml / minute was aqueous feed. About 10 ml of the aqueous stock solution was processed. The resulting suspension was simultaneously fed into a 0.4-micron filter to collect the particles. The particles were then washed with pentanol while on the filter and dried under nitrogen. The powder contained 1.1% residual water, 0.1% residual pentanol, and 0.5% residual octanol by weight. A sample of the unfiltered suspension was retained for particle size analysis. 11,321 particles were imaged for particle size analysis. The average particle size was 2.5 microns, with the largest particle measuring 8.5 microns.

[0143] For the second batch, a protein stock solution was prepared at a total solids concentration of about 102 mg / ml. The octanol and aqueous stock solutions were fed into a tandem homogenizer (IKA T-25) with custom feeds at speeds of 156 and 1.963 ml / min, respectively. About 99 ml of the aqueous stock solution was processed. The resulting suspension was then fed into a 0.4-micron filter to collect the particles. The filtration was performed at 10 atmospheres of pressure supplied by compressed nitrogen gas. The particles were then washed with pentanol while on the filter and dried under nitrogen. The powder contained 0.9% residual water, 0.1% residual pentanol, and 0.7% residual octanol by weight. A sample of the unfiltered suspension was retained for particle size analysis. 13,135 particles were imaged for particle size analysis. The average particle size was 2.4 microns, with the largest particle measured at 9.9 microns.

[0144] For the third batch, a protein stock solution was prepared at a total solids concentration of about 103 mg / ml. The octanol and aqueous stock solutions were fed into a tandem homogenizer (IKA T-25) with custom feeds at speeds of 155 and 1.946 ml / min, respectively. About 110 ml of the aqueous stock solution was processed. The resulting suspension was then fed into a 0.4-micron filter to collect the particles. The filtration was performed at 10 atmospheres of pressure supplied by compressed nitrogen gas. The particles were then washed with pentanol while on the filter and dried under nitrogen. The powder contained 1.5% residual water, 0.1% residual pentanol, and 0.7% residual octanol by weight. A sample of the unfiltered suspension was retained for particle size analysis. 12,816 particles were imaged for particle size analysis. The average particle size was 2.6 microns, with the largest particle measured at 10.1 microns.

[0145] Example 3 In vitro release of a suspension of micro-glassy protein particles into release media comprising 0.25 mg / mL hyaluronidase in phosphate buffered saline (PBS) (PBS-Hyal media) or PBS containing 0.5% hyaluronic acid and hyaluronidase at a concentration of 0.25 mg / mL (PBS-HA-Hyal media) was tested and compared. A release media of PBS and 0.25 mg / mL hyaluronidase was also included as a control. The suspension of particles contained bovine gamma globulin (BGG) and sucrose and was prepared in benzyl benzoate at a concentration of 500 mg / mL total solids. About 10 μΐ^of the suspension was added to 5 mL tubes containing either PBS-Hyal release media or PBS-HA-Hyal release media.

[0146] The release media containing the suspension of particles was sampled over time and the absorbance of the sampled release media at 280 nm was measured to determine the release of BGG from the suspension over time (e.g., the concentration of BGG released from the particles and / or suspension and / or dissolved in the release media over time after contact with the release media). As seen from Figure 3 As can be seen, the samples containing the release media PBS-HA show a slow release of BGG from the suspension over time, while the samples containing PBS-HA-Hyal release media show a rapid release of BGG from the suspension over time. The results of the experiment demonstrate the feasibility of using hyaluronidase in an aqueous phase to increase the rate of dissolution of a solidified protein, such as from a non-aqueous suspension, in a subcutaneous space. Figure 1 As can be seen, the samples containing the release media PBS-HA show a slow release of BGG from the suspension over time, while the samples containing PBS-HA-Hyal release media show a rapid release of BGG from the suspension over time. The results of the experiment demonstrate the feasibility of using hyaluronidase in an aqueous phase to increase the rate of dissolution of a solidified protein, such as from a non-aqueous suspension, in a subcutaneous space.

[0147] Example 4 For three independent samples, the in vitro release of a suspension of protein particles into a solution of PBS (PBS media-1, PBS media-2, or PBS media-3) or 0.5% hyaluronic acid (HA) in PBS (PBS-HA media-1, PBS-HA media-2, or PBS HA media-3) was tested and compared. The in vitro release of a suspension of protein particles in PBS or HA was also compared to the in vitro release of a suspension containing particles comprising the same protein and hyaluronidase in PBS or HA.

[0148] A suspension of micro-glassized particles containing bovine gamma globulin (BGG) and sucrose was prepared in benzyl benzoate at a concentration of 500 mg / mL total solids. About 10 μL of the suspension was added to a 5 mL tube with PBS or HA as the release media. The release media containing the suspension of BGG particles was sampled over time and the absorbance of the sampled release media at 280 nm was measured to determine the concentration of protein in the release media over time. Triplicate samples were prepared for each release media used.

[0149] Figure 2 The measured absorbance over time is shown for each of the three PBS media samples, and Figure 3 The measured absorbance over time is shown for each of the three PBS-HA media samples. In Figure 2 and Figure 3 The maximum absorbance measured indicates the time at which the protein (BGG) is completely released from the suspension. As Figure 2The suspension added to the PBS medium showed rapid release of protein over time, with complete release of protein within one hour, as shown in FIG. 1. The suspension added to the 0.5% hyaluronic acid (HA) release medium showed slow release, with complete release of protein occurring over about a day, as shown in FIG. 2. Figure 3

[0150] A suspension of micro-glassy particles comprising hyaluronidase and BGG was prepared from a solution comprising hyaluronidase in an amount of 2% by mass of dissolved solids and BGG in an amount of about 98% by mass of dissolved solids. The particles were provided in the suspension in benzyl benzoate at a concentration of about 500 mg / mL total solids. The same experiment as described above was performed, wherein about 10 μL of the suspension comprising particles containing BGG and hyaluronidase was added to a 5 mL tube with either PBS or HA as the release medium. Assuming complete release of hyaluronidase from the particles, there would be 0.025 mg of hyaluronidase per mL of release medium, which is 1 / 10 the amount of hyaluronidase in Example 3. The release medium was sampled over time in the same manner as described above, wherein the absorbance of the sampled release medium was measured at 280 nm to determine the protein concentration in the release medium over time. Triplicate samples were prepared for each release medium used.

[0151] Figure 4 The measured absorbance of each PBS sample (PBS Medium-1, PBS Medium-2, or PBS Medium-3) comprising particles containing BGG and hyaluronidase is shown over time, and Figure 5 The measured absorbance of each of the three PBS-HA medium samples (PBS-HA Medium-1, PBS-HA Medium-2, or PBS HA Medium-3) comprising particles containing BGG and hyaluronidase is shown over time. In Figure 4 and Figure 5 The measured maximum absorbance indicates the time at which the protein is completely released from the suspension, as shown in Figure 4 and Figure 5 For both the PBS release medium and the PBS-HA release medium, the protein completed release from the suspension within one hour, as shown in FIGS. 3 and 4, respectively. These results indicate that the hyaluronidase contained in the solid protein particles is able to effectively break down the hyaluronic acid in the HA release medium and allow the protein to dissolve more rapidly from the suspension into the aqueous phase of the release medium. Furthermore, the amount of hyaluronidase in the particles prepared in Example 4 is about 1 / 25 the amount of hyaluronidase added to the medium in Example 3, indicating that a smaller amount of hyaluronidase in the form of particles is needed to increase the release of protein from the suspension compared to free hyaluronidase in solution.

[0152] Example 5 ​ Protein release from suspensions containing solid particles with varying amounts of hyaluronidase will be compared. Particles with varying amounts of hyaluronidase in the particles will be prepared (e.g., amounts of hyaluronidase of 0.1%, 0.5%, 1%, 3%, or 4% by weight of the particles) and can be prepared as described in one or more of Examples 1-4.

[0153] Example 6 Protein release from suspensions of hyaluronidase-containing particles of the application and separate solid protein particles will be compared. Particles with varying amounts of hyaluronidase in the particles will be prepared (e.g., amounts of hyaluronidase of 0.1%, 0.5%, 1%, 3%, or 4% by weight of the particles) and can be prepared as described in one or more of Examples 1-4. Hyaluronidase particles will be provided in suspensions with particles that do not contain hyaluronidase and suspensions with varying concentrations of hyaluronidase particles will be prepared. For example, suspensions containing 0.1%, 0.5%, 1%, 3%, or 4% of the hyaluronidase particles present by weight of the total solid content of the suspension and suspensions containing particles that do not contain hyaluronidase can be prepared.

[0154] Example 7 Enzymatic activity of the solid protein particles containing hyaluronidase will be measured over time (e.g., using Dorfman, A. (1955) Methods in Enzymology, Volume I, 166-173 for hyaluronidase EC 3.2.1.35, HAse, turbidity measurement and comparing turbidity to a standard curve) and compared to the activity of the hyaluronidase in the stock formulation. Particles containing hyaluronidase are prepared in formulations as shown in Table 1.

[0155] Table 1: Hyaluronidase particle formulation. Formulation ID Sugar Type Amino Acid (AA) Hyaluronidase: Sugar: AA: Buffer F1 NA NA 98:0:0:2 F2 Trehalose NA 60:38:0:2 F3 Sucrose NA 60:38:0:2 F4 Sucrose Proline 40:29:29:2

[0156] Powder of the particle formulation will be placed at 2-8°C or 25°C for 3 months for stability. At predetermined time points (initially (t=0), after 30 days, and after 90 days), the powder will be rehydrated (using 20 mM sodium phosphate pH 7.0 at 37°C with 77 mM sodium chloride and 0.01% (w / v) bovine serum albumin) and the activity of the enzyme measured as described in Dorfman, A. (1955) Methods in Enzymology, Volume I, 166-173. Results are shown in Figure 6 and Figure 7 .

[0157] Example 8 Using Gottingen A minipig model will compare injection site reactions and pharmacokinetics of injected suspensions with and without hyaluronidase particles of the invention in non-aqueous vehicles and injected suspensions containing micro-glassy particles comprising a therapeutic agent with or without hyaluronidase.

[0158] The formulations were prepared according to the following table (BB = benzyl benzoate; EO = ethyl oleate): The animals used were male Gottingen minipigs, 7-8 months old. 2 ml of each formulation was injected into the flank using a 27G needle over ~10 s (4 groups of 4 animals each, for a total of 8 animals). Immediately after injection, at 1, 2, 3, 4 and 6 hours after injection and once daily for 14 days, the injection sites were scored for local changes (heat, redness, swelling, blister size).

[0159] Example 9 Dry, dense particles containing hyaluronidase, sucrose and bovine serum albumin (BSA) were produced using the micro-glassy method and compared to particles of BSA without hyaluronidase. Two batches were produced. The resulting particles with enzyme contained about 0.37% hyaluronidase by weight of the particles.

[0160] For the first batch, a stock solution of BSA and sucrose was prepared at a total solids concentration of about 85 mg / ml. Octanol and aqueous stock were fed into a tandem homogenizer (IKA T-25) to provide discrete, spherical, solid particles. About 33 ml of the aqueous stock was processed. The resulting suspension was washed with solvent, filtered and dried under nitrogen. The powder contained 3.2% residual water, 0.3% residual wash solvent and 5.2% residual octanol by weight. A sample of the unfiltered suspension was retained for particle size analysis. 8,559 particles were imaged for particle size analysis. The average particle size was 2.7 microns, with the largest particle measured at 11.9 microns.

[0161] For the second batch, a stock solution of BSA, sucrose, and hyaluronidase was prepared at a total solids concentration of about 85 mg / ml. Octanol and the aqueous stock solution were fed into a tandem homogenizer (IKA T-25) to provide discrete, spherical, solid particles. About 33 ml of the aqueous stock solution was processed. The resulting suspension was washed with solvent, filtered, and dried under nitrogen. The powder contained 4.2% residual moisture, 0.2% residual washing solvent, and 5.3% residual octanol by weight. A sample of the unfiltered suspension was retained for particle size analysis. 12,583 particles were imaged for particle size analysis. The average particle size was 2.5 microns, with the largest particle measured at 10.7 microns.

[0162] The powder was placed at 2-8°C, 25°C, and 40°C for 1 month for stability. At predetermined time points, the powder was rehydrated, enzyme activity was measured as described in Dorfman, A. (1955) Methods in Enzymology, Vol. I, 166-173, and BSA aggregation was measured by SEC-HPLC. Figure 8 Figure 9

[0163] Example 10 Dry, dense particles containing hyaluronidase, trehalose, and a monoclonal antibody (mAb) were produced using the micro-glassification method and compared to particles of the mAb without hyaluronidase. Two batches were produced. The resulting particles with enzyme contained about 0.3% hyaluronidase by weight of the particles.

[0164] For the first batch, a stock solution of the mAb and trehalose was prepared at a total solids concentration of about 101 mg / ml. Octanol and the aqueous stock solution were fed into a tandem homogenizer (IKA T-25) to provide discrete, spherical, solid particles. About 10 ml of the aqueous stock solution was processed. The resulting suspension was washed with solvent, filtered, and dried under nitrogen. The powder contained 1.8% residual moisture, 0.1% residual washing solvent, and 0.5% residual octanol by weight. A sample of the unfiltered suspension was retained for particle size analysis. 12,779 particles were imaged for particle size analysis. The average particle size was 2.6 microns, with the largest particle measured at 8.9 microns.

[0165] ​​For the second batch, a stock solution of mAb, hyaluronidase and trehalose was prepared at a total solids concentration of approximately 100 mg / ml. The octanol and aqueous stock solutions were fed into an inline homogenizer (IKA T-25) to provide discrete, spherical, solid particles. Approximately 10 ml of the aqueous stock solution was processed. The resulting suspension was washed with solvent, filtered and dried under nitrogen. The powder contained 0.6% residual moisture, 0.1% residual washing solvent and 0.4% residual octanol by weight. A sample of the unfiltered suspension was retained for particle size analysis. 11,742 particles were imaged for particle size analysis. The average particle size was 2.7 microns, with the largest particle measured at 16.1 microns ( Figure 10 ).

[0166] The powder was placed for stability at 2-8°C, 25°C and 40°C for 60 days. On days 0, 15 and 30, the powder was rehydrated and the monomer percentage of mAb was measured via SEC-HPLC ( Figure 11 、 Figure 12 、 Figure 13 、 Figure 14 、 Figure 15 and Figure 16 ), and enzyme activity was measured as described in Dorfman, A. (1955) Methods in Enzymology, Vol. 1, 166-173 ( Figure 17 ).

[0167] Example 11 In contrast to Example 10, microbatch microvitrification was used to form particles, with approximately 12 μl of aqueous stock solution processed for each sample. Granule formulations containing mAb, hyaluronidase, and various excipients were formed (Table 2) and stored at 40°C for stability. After 12 days, the powder was rehydrated, enzyme activity was determined as described in Dorfman, A. (1955) Methods in Enzymology, Vol. 1, 166-173, and mAb aggregation was measured by SEC-HPLC (Table 3).

[0168] Table 2: Microbatches of additional hyaluronidase preparations a = 10 mM phosphate, pH 7.2, b = phosphate buffered saline, c = 10 mM histidine, pH 6.0 Table 3: Hyaluronidase (HYAL) activity of microbatches (F5 to F7 and F9 to F11) and change in percentage monomer in mAb (for microbatches F7 to F11) after storage for 12 days at 40°C & Data unavailable Example 12 Nanoparticles of hyaluronidase (e.g., particles having an average size of about 100 nm to about 500 nm) will be produced using the above method by adding an aqueous feed comprising about 1-5 mg / ml hyaluronidase to a dehydrating solvent. Activity of the powder will be determined using one of the above methods.

[0169] The foregoing is considered as illustrative only of the principles of the application. Further, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the application to the exact construction and operation described. Accordingly, all such variations are intended to be included within the scope of the present application as defined in the claims below and the equivalents thereof. The disclosures of all articles and publications referred to herein are incorporated herein by reference in their entirety.

Claims

1. A particle comprising: Hyaluronidase; and therapeutic agents, wherein the particles have a water content of less than about 15% by weight of the particles and / or a water activity of less than about 0.

9.

2. The particle of claim 1, wherein the therapeutic agent is a biologic, optionally wherein the biologic is selected from the group consisting of amino acids, peptides, proteins, nucleotides, polynucleotides, and any combination thereof.

3. The particle of claim 1, wherein the therapeutic agent is a small molecule (e.g., a small organic molecule).

4. The particle according to any preceding claim, further comprising a stabilizer, optionally wherein the stabilizer is a sugar (e.g., sucrose, glucose, trehalose, mannitol, sorbitol, dextrose, maltose and / or lactose), an amino acid and / or a protein (e.g., an albumin, such as serum albumin).

5. The particle of claim 4, wherein the stabilizer is present in the particle in an amount from about 0.1%, 5%, 10%, 15% or 20% to about 25%, 30%, 35%, 40%, 45% or 50% by weight of the particle.

6. The particle of any one of claims 4 or 5, wherein the stabilizer is not covalently bound to the hyaluronidase.

7. The particle according to any one of claims 4 to 6, wherein the stabilizer is an amino acid, optionally wherein the amino acid is selected from the group consisting of histidine, arginine, proline, glycine, leucine, and any combination thereof.

8. The particle of any one of claims 4-7, wherein the stabilizer is present in the particle in an amount of about 5% to about 30% by weight of the particle.

9. The particle of any preceding claim, wherein the hyaluronidase is present in the particle in an amount from about 0.1%, 0.5%, or 1% to about 2%, 3%, 4%, or 5% by weight of the particle.

10. The particle of any preceding claim, wherein the therapeutic agent is present in the particle in an amount from about 50%, 55%, 60%, 65% or 70% to about 75%, 80%, 85%, 90%, 95% or 99% by weight of the particle, optionally wherein the therapeutic agent is present in the particle in an amount from about 75% or 80% to about 85%, 90% or 95% by weight of the particle.

11. The particle of any preceding claim, wherein the hyaluronidase and the therapeutic agent are not covalently bound.

12. A particle comprising: Hyaluronidase; and stabilizers, wherein the particles have a water content of less than about 15% by weight of the particles and / or a water activity of less than about 0.

9.

13. The particle of claim 12, wherein the stabilizer is a sugar (e.g., sucrose, glucose, trehalose, mannitol, sorbitol, dextrose, maltose and / or lactose), an amino acid and / or a protein (e.g., albumin, such as serum albumin), optionally wherein the sugar is selected from sucrose, glucose, trehalose, mannitol, sorbitol, dextrose, maltose, lactose, and any combination thereof.

14. The particle of claim 12, wherein the stabilizer is an amino acid, optionally wherein the amino acid is selected from the group consisting of histidine, arginine, proline, glycine, leucine, and any combination thereof.

15. The particle of claim 12, wherein the stabilizer is albumin, optionally serum albumin.

16. The particle of any one of claims 12-15, wherein the stabilizer is present in the particle in an amount from about 0.1%, 0.5%, 1%, 5%, 10%, 15% or 20% to about 25%, 30%, 35%, 40%, 45% or 50% by weight of the particle.

17. The particle of any one of claims 12-16, wherein the hyaluronidase and the stabilizer are not covalently bound.

18. The particle of any one of claims 12-18, wherein the hyaluronidase is present in the particle in an amount from about 50%, 55%, 60%, 65% or 70% to about 75%, 80%, 85%, 90%, 95% or 99% by weight of the particle.

19. A particle according to any preceding claim, wherein the particle is spherical in shape.

20. The particle of any preceding claim, wherein the particle has a g / cm 2 of at least about 0.5, 0.6, 0.7, 0.8, 0.9, or 1 g / cm 2. 3 to about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 g / cm 3 density.

21. The particle of any preceding claim, wherein the particle has a size (e.g., diameter) of about 0.1 microns to about 30 microns, optionally wherein the particle has a size of about 2 microns to about 5 microns, about 5 microns to about 20 microns, or about 10 microns to about 30 microns.

22. The particle of any preceding claim, wherein the particle has a total moisture content of less than about 3% of the total particle mass, optionally wherein the particle has a total moisture content of less than about 2% of the total particle mass.

23. A particle according to any preceding claim, wherein the particle has a residual (e.g. octanol and / or pentanol) content in an amount less than about 3% by weight of the total particle mass.

24. The particle of any preceding claim, wherein the particle is amorphous and / or wherein the hyaluronidase in the particle is amorphous.

25. The particle of any preceding claim, wherein the particle is a microparticle or a nanoparticle.

26. The particle of any preceding claim, wherein the enzymatic activity of the hyaluronidase after dissolution of the particle in an aqueous composition is within about ±50% of the enzymatic activity of the free hyaluronidase, optionally wherein the enzymatic activity of the hyaluronidase after dissolution of the particle in an aqueous composition is within about ±40% of the enzymatic activity of the free hyaluronidase.

27. The particle of any preceding claim, wherein the enzymatic activity of the hyaluronidase is within about ±50% of the enzymatic activity of the free hyaluronidase after storage for at least about 3 months at about 4°C to about 40°C, optionally wherein the enzymatic activity of the hyaluronidase is within about ±40% of the enzymatic activity of the free hyaluronidase after storage for at least about 3 months at about 4°C to about 40°C.

28. The particle of any preceding claim, wherein the hyaluronidase is hyaluronidase V.

29. A composition comprising particles according to any preceding claim.

30. The composition of claim 29, wherein the composition is a suspension.

31. The composition of claim 29 or 30, wherein the reaction mixture is heated to a temperature of about 20 degrees Celsius to about 25 degrees Celsius and at a temperature of about 1,000 s -1 to about 3,000s -1 The composition has a viscosity of about 20 centipoise (cP) to about 200 cP when measured at a shear rate of 100 Å.

32. The composition of any one of claims 29-31, wherein the particles are present in the composition in an amount from about 1, 10, 50, 100, or 200 mg / mL to about 300, 400, 500, 600, or 700 mg / mL.

33. The composition of any one of claims 29-32, wherein the composition is non-aqueous.

34. The composition of any one of claims 29-33, further comprising a solvent, optionally wherein the solvent is an ester (eg, an alkyl ester or an aryl ester).

35. The composition according to claim 34, wherein the solvent is selected from benzyl benzoate, ethyl oleate, triacylglycerol (e.g., 812), ethyl lactate, sesame oil and any combination thereof.

36. The composition of any one of claims 29-35, wherein the hyaluronidase is insoluble in the solvent and / or composition.

37. The composition of any one of claims 29-36, wherein the hyaluronidase is in solid form in the composition.

38. The composition of any one of claims 29-37, further comprising a therapeutic agent dissolved or suspended in the composition.

39. A composition comprising: solvent; and A particle comprising a hyaluronidase, wherein the particle has a water content of less than about 15% by weight of the particle and / or a water activity of less than about 0.

9.

40. The composition of claim 39, further comprising a therapeutic agent, optionally wherein the therapeutic agent is dissolved or suspended in the composition.

41. The composition of claim 39 or 40, wherein the composition is non-aqueous.

42. The composition of any one of claims 39-41, wherein the hyaluronidase is insoluble in the solvent and / or composition.

43. The composition of any one of claims 39-42, wherein the hyaluronidase is in solid form in the composition.

44. The composition of any one of claims 39-43, wherein the composition is a suspension.

45. according to the composition of any one of claims 39-44, wherein it is equivalent to the temperature of about 20 degrees Celsius to about 25 degrees Celsius and about 1,000s -1 to about 3,000s -1 The composition has a viscosity of about 20 centipoise (cP) to about 200 cP when measured at a shear rate of 100 Å.

46. ​​The composition of any one of claims 39-45, wherein the particles are present in the composition in an amount from about 1, 10, 50, 100, or 200 mg / mL to about 300, 400, 500, 600, or 700 mg / mL.

47. The composition of any one of claims 29-46, further comprising a detergent (e.g., polysorbate-20, polysorbate-80, and / or poloxamer 188).

48. A method of increasing the dissolution of a therapeutic agent in a liquid, the method comprising: The liquid, the therapeutic agent, and particles comprising a hyaluronidase are combined, wherein the particles have a water content of less than about 15% by weight of the particles and / or a water activity of less than about 0.9, thereby increasing the dissolution of the therapeutic agent in the liquid.

49. The method of claim 48, wherein the therapeutic agent is separated from the particles, optionally wherein the therapeutic agent and / or the particles are present in a solvent different from the liquid.

50. The method of claim 48, wherein the particles comprise a therapeutic agent, optionally wherein the particles are present in a solvent different from the liquid.

51. The method of claim 48, wherein the particle is a particle according to any one of claims 1-28.

52. The method of claim 48, wherein the particles are present in a composition, optionally wherein the composition is a composition according to any one of claims 29-47.

53. The method of any one of claims 48-52, wherein the liquid is an aqueous liquid.

54. The method of any one of claims 48-53, wherein the combining comprises separately combining the hyaluronidase and the therapeutic agent with the liquid, optionally wherein the hyaluronidase and the therapeutic agent are combined with the liquid (e.g., added to the liquid) sequentially.

55. The method of any one of claims 48-53, wherein the combining comprises simultaneously combining the hyaluronidase and the therapeutic agent in the liquid, optionally wherein the hyaluronidase and the therapeutic agent are present in the same particles or in different particles.

56. The method of any one of claims 48-55, wherein dissolution of the therapeutic agent is increased by at least about 10%, 20%, 30%, 40%, 50%, 60% or more over a period of about 5 minutes to about 2 hours compared to dissolution of the therapeutic agent in the absence of the particles.

57. The method of any one of claims 48-56, wherein at least about 90%, 95%, 99% or 100% of the therapeutic agent dissolves within about 20, 30 or 40 minutes to about 100, 110 or 120 minutes after combining the particles, therapeutic agent and the liquid, optionally wherein at least about 90%, 95%, 99% or 100% of the therapeutic agent dissolves within about 20, 30 or 40 minutes to about 50, 60, 70 or 80 minutes after combining the particles, therapeutic agent and the liquid.

58. A method of improving the tolerability of and / or increasing the absorption of a therapeutic agent when administered subcutaneously, intramuscularly, and / or intradermally to a subject, the method comprising: administering particles comprising a hyaluronidase to the subject subcutaneously, intramuscularly, and / or intradermally, wherein the particles have a water content of less than about 15% by weight of the particles and / or a water activity of less than about 0.9, thereby improving tolerability of the therapeutic agent and / or increasing absorption of the therapeutic agent when administered subcutaneously, intramuscularly, and / or intradermally to the subject.

59. The method of claim 58, wherein the therapeutic agent is separated from the particles.

60. The method of claim 58, wherein the particles comprise the therapeutic agent.

61. The method of claim 58, wherein the particle is a particle according to any one of claims 1-28.

62. The method of claim 58, wherein the subcutaneous, intramuscular and / or intradermal administration comprises administering a composition comprising the particles subcutaneously, intramuscularly and / or intradermally to the subject, optionally wherein the composition is a composition according to any one of claims 29-47.

63. The method of any one of claims 58-62, wherein the method reduces irritation to the subject at an injection site for subcutaneous, intramuscular, and / or intradermal administration.

64. The method of any one of claims 58-63, wherein the method increases dispersion of the particles or composition beneath the subject's skin following the subcutaneous, intramuscular, and / or intradermal administration.

65. The method of any one of claims 58-64, wherein the method increases absorption, bioavailability, distribution, metabolism, and / or excretion of the therapeutic agent in the subject following subcutaneous, intramuscular, and / or intradermal administration as compared to absorption, bioavailability, distribution, metabolism, and / or excretion of the therapeutic agent in the subject following subcutaneous, intramuscular, and / or intradermal administration in the absence of the particle or composition.

66. The method of any one of claims 58-65, wherein the subcutaneous, intramuscular, and / or intradermal administration comprises administering the particle or composition to the subject subcutaneously, intramuscularly, and / or intradermally in a volume of about 0.1 or 0.5 mL to about 1, 2, 5, or 10 mL.

67. The method of any one of claims 58-66, wherein the method does not administer hyaluronidase in solution (e.g., hyaluronidase dissolved in water) and / or free hyaluronidase.

68. The method of any one of claims 58-67, wherein the particles and / or hyaluronidase are in solid form when administered subcutaneously, intramuscularly, and / or intradermally to the subject, optionally wherein the hyaluronidase in the particles dissolves upon subcutaneous, intramuscular, and / or intradermal administration.

69. The method of any one of claims 58-68, wherein the subcutaneous, intramuscular, and / or intradermal administration comprises separate subcutaneous, intramuscular, and / or intradermal administration of the hyaluronidase and the therapeutic agent.

70. The method of claim 69, wherein the subcutaneous, intramuscular, and / or intradermal administration comprises administering the hyaluronidase to the subject subcutaneously, intramuscularly, and / or intradermally, and then administering the therapeutic agent to the subject subcutaneously, intramuscularly, and / or intradermally.

71. The method of claim 69, wherein the subcutaneous, intramuscular, and / or intradermal administration comprises administering the therapeutic agent to the subject subcutaneously, intramuscularly, and / or intradermally, and then administering the hyaluronidase to the subject subcutaneously, intramuscularly, and / or intradermally.

72. The method of any one of claims 58-68, wherein the subcutaneous, intramuscular, and / or intradermal administration comprises administering the hyaluronidase and the therapeutic agent to the subject simultaneously, optionally wherein the hyaluronidase and the therapeutic agent are present in the same particles or the same composition.

73. The method of any one of claims 58-72, wherein at least about 90%, 95%, 99% or 100% of the therapeutic agent dissolves within about 20, 30 or 40 minutes to about 100, 110 or 120 minutes following subcutaneous, intramuscular and / or intradermal administration to the subject, optionally wherein at least about 90%, 95%, 99% or 100% of the therapeutic agent dissolves within about 20, 30 or 40 minutes to about 50, 60, 70 or 80 minutes following subcutaneous, intramuscular and / or intradermal administration to the subject.

74. The method of any one of claims 58-73, wherein the hyaluronidase is administered to the subject in an amount of about 0.1%, 0.5%, or 1% to about 2%, 3%, 4%, or 5% by weight of the particle.

75. The method of any one of claims 58-74, wherein the subcutaneous, intramuscular, and / or intradermal administration comprises administering the particles and therapeutic agent in a total volume of less than about 10 mL, optionally in a total volume of less than about 2 mL.

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