Hyaluronidase particles, compositions containing 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 present invention may comprise hyaluronidase particles and may be suspensions. The hyaluronidase-containing particles may 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. In some embodiments, the methods of the present invention may include administering hyaluronidase-containing particles subcutaneously, intramuscularly, and / or intradermally to a subject, 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.

Description

[Technical Field]

[0001] 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 by reference in its entirety.

[0002] Statement Regarding Electronic Filing of Sequence Listings The XML sequence listing, file name "1458-5WO_ST26.xml", size 1,557,299 bytes, created on February 19, 2024, and attached to this application, is incorporated herein by reference in its entirety.

[0003] [Technical field] The present invention relates to hyaluronidase particles, compositions comprising the hyaluronidase particles, and methods of making and using the hyaluronidase particles. [Background technology]

[0004] Subcutaneous injections offer a flexible and effective route of drug administration, in which pharmaceuticals are injected into the tissue layer between the skin and muscle. Subcutaneous injections are advantageous in that they are an inexpensive and painless method of parenteral administration with a low risk of systemic infection and a slow absorption rate, thereby providing long-term efficacy of the injected drug. However, fluids injected subcutaneously remain largely at the injection site and slowly disappear over several hours to several days. Injecting large volumes (e.g., more than about 2 mL) can result in fluid retention and / or swelling at the injection site, which can cause local side effects such as pain, irritation, and / or skin damage. For this reason, subcutaneous injections are typically limited to small doses, e.g., volumes less than about 2 mL.

[0005] Hyaluronidase is a family of enzymes, particularly endoglycosidases, that can degrade hyaluronic acid and, in some cases, other glycoproteins, such as chondroitin and chondroitin sulfate. Hyaluronidases are present in organs, such as the testes, spleen, and liver, and in body fluids, such as tears and blood. Hyaluronidases exist in three distinct classes: mammalian (or vertebrate), bacterial, and leech / bloodworm. Within the mammalian class, the human genome contains six known genes encoding hyaluronidase-like sequences. Purified human hyaluronidases, such as testicular hyaluronidase PH-20 or ovine hyaluronidase, such as ovine PH-20, 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 degrading the hyaluronic acid network in the subcutaneous space, subcutaneous administration of hyaluronidase in aqueous solution can disperse subcutaneously injected fluids more quickly than in the absence of hyaluronidase, thereby allowing large volumes of fluid to be administered. Hyaluronidase in aqueous solution has been approved by the US Food and Drug Administration (FDA) for use as an adjuvant in the rapid administration of large volumes of fluids of 1,000 mL or more or in the subcutaneous administration of other drugs. Hyaluronidase is typically administered as an aqueous solution before injecting a large volume of drug greater than 2 mL, or as a coformulation with a large volume of drug greater than 2 mL. Summary of the Invention [Problem to be solved by the invention]

[0007] Novel approaches for the administration of hyaluronidase and therapeutic agents are needed, particularly for subcutaneous and intradermal administration. [Means for solving the problem]

[0008] A first aspect of the present invention is directed to particles comprising hyaluronidase, wherein the particles 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.

[0009] A further aspect of the present invention is directed to particles comprising hyaluronidase and a therapeutic agent, wherein the particles 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.

[0010] Another aspect of the present invention is directed to particles comprising hyaluronidase and a stabilizer, 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.

[0011] A further aspect of the present invention is directed to a composition comprising the particles described herein.

[0012] Another aspect of the present invention is directed to a composition comprising a solvent and particles comprising hyaluronidase, 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.

[0013] A further aspect of the present invention is directed to a method for increasing the dissolution of a therapeutic agent in a liquid, the method comprising combining the liquid, the therapeutic agent, and particles comprising hyaluronidase, the particles having 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.

[0014] Another aspect of the present invention is directed to a method of improving tolerance and / or increasing absorption of a therapeutic agent upon subcutaneous, intramuscular, and / or intradermal administration to a subject, comprising subcutaneously, intramuscularly, and / or intradermally administering to the subject particles comprising hyaluronidase, the particles having 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 tolerance and / or increasing absorption of the therapeutic agent upon subcutaneous, intramuscular, and / or intradermal administration to the subject. In some embodiments, the therapeutic agent is administered subcutaneously, intramuscularly, and / or intradermally to the subject before, during, and / or after subcutaneously, intramuscularly, and / or intradermally administering the particles to the subject.

[0015] It should be noted that aspects of the invention described herein with respect to one embodiment may be incorporated into a different embodiment, even if not specifically described therein. That is, all embodiments and / or features of any embodiment may be combined in any manner and / or combination. Applicant reserves the right to amend the originally filed claims and / or to submit any new claims as appropriate, including the right to make the originally filed claims rely on and / or incorporate other claims or claim features not originally asserted. These and other objects and / or features of the present invention are described in detail in the specification set forth below. Further features, advantages, and details of the present invention will be apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments. Such description is merely exemplary of the invention. [Brief explanation of the drawings]

[0016] [Figure 1]FIG. 1 is a graph of absorbance at 280 nm over time (in hours) showing the amount of bovine gamma globulin (BGG) released from a benzyl benzoate suspension into a medium containing phosphate buffered saline (PBS) and 0.25 mg / mL hyaluronidase (PBS-Hyal medium) or into a medium containing PBS, 0.5% hyaluronic acid, and 0.25 mg / mL hyaluronidase (PBS-HA-Hyal medium). [Figure 2] FIG. 2 is a graph of absorbance at 280 nm over time (in hours) showing the amount of BGG released from benzyl benzoate suspensions into PBS for three separate samples (i.e., PBS medium-1, PBS medium-2, and PBS medium-3). [Figure 3] FIG. 3 is a graph of absorbance at 280 nm over time (in hours) showing the amount of BGG released from benzyl benzoate suspension into a medium containing 0.5% hyaluronic acid (HA) and PBS for three separate samples (i.e., PBS-HA medium-1, PBS-HA medium-2, and PBS-HA medium-3). [Figure 4] FIG. 4 is a graph of absorbance at 280 nm over time (in hours) showing the amount of BGG released into PBS from a benzyl benzoate suspension for three separate samples (i.e., PBS medium-1, PBS medium-2, and PBS medium-3), wherein the suspension comprises a plurality of particles according to some embodiments of the present invention, each of the plurality of particles comprising BGG and hyaluronidase. [Figure 5] FIG. 5 is a graph of absorbance at 280 nm over time (in hours) showing the amount of BGG released from a benzyl benzoate suspension into a medium containing 0.5% hyaluronic acid (HA) and PBS, wherein the suspension contains a plurality of particles according to some embodiments of the present invention, each of the plurality of particles containing BGG and hyaluronidase. [Figure 6]FIG. 6 is a graph showing the stability of microbatches of various hyaluronidase powder formulations stored at 25° C., in terms of enzyme activity compared to the activity level of the stock solution before storage (i.e., at the time of initial formation of the stock solution, t=0). [Figure 7] FIG. 7 is a graph showing the stability of microbatches of various hyaluronidase powder formulations stored at 2-8° C., in terms of enzyme activity compared to the activity level of the stock solution prior to storage. [Figure 8] FIG. 8 is a graph of the activity of hyaluronidase over time when stored at the indicated temperatures in a powder formulation containing the enzyme and bovine serum albumin (BSA). [Figure 9] FIG. 9 is a graph of the change in the percentage of BSA monomer over time in powders containing only BSA or in powders containing a combination of BSA and hyaluronidase. [Figure 10] FIG. 10 is a microscopic image showing the spherical morphology of particles of monoclonal antibody (mAb) and approximately 0.3% hyaluronidase suspended in octanol. [Figure 11] FIG. 11 is a graph of mAb stability, as indicated by monomer percentage, from particles containing mAb only or from particles containing mAb and hyaluronidase when stored at 2-8° C. [Figure 12] FIG. 12 is a graph of mAb stability, as indicated by the percentage of high-molecular weight species (HMWS) from particles containing only mAb or from particles containing a combination of mAb and hyaluronidase, when stored at 2-8°C. [Figure 13] FIG. 13 is a graph of mAb stability, as indicated by percentage monomer, from particles containing only mAb or from particles containing a combination of mAb and hyaluronidase when stored at 25° C. [Figure 14]FIG. 14 is a graph of the percentage of HMWS from particles containing only mAb or from particles containing a combination of mAb and hyaluronidase when stored at 25° C. [Figure 15] FIG. 15 is a graph of mAb stability, as indicated by percentage monomer, from particles containing only mAb or from particles containing a combination of mAb and hyaluronidase when stored at 40° C. [Figure 16] FIG. 16 is a graph of the percentage of HMWS from particles containing only mAb or from particles containing a combination of mAb and hyaluronidase when stored at 40° C. [Figure 17] Figure 17 is a graph of hyaluronidase activity over time in particles containing mAb alone or in particles containing a combination of mAb and hyaluronidase when stored at 2-8, 25 or 40°C. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention is described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. However, the present invention may 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 will fully convey the scope of the invention to those skilled in the art.

[0018] The terms used in the detailed description of the invention herein are for the purpose of describing particular embodiments only and are not intended to be limiting of the invention. As used in the detailed description of the invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0019] Although the terms "first," "second," etc. may be used herein to describe various elements, it will be understood that these elements should not be limited by these terms. These terms are used only to distinguish one element from another. Thus, a "first" element could be referred to as a "second" element without departing from the teachings of the present embodiment. The order of procedures (or steps) presented in the claims or drawings is not necessarily limited to that order unless specifically stated.

[0020] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Furthermore, unless explicitly defined herein, words as defined in commonly used dictionaries should be interpreted to have a meaning consistent with their meaning in the context of this application and the related art, and should not be interpreted in an idealized or overly formal sense. The terms used in the description of this specification are intended only to describe particular embodiments and are not intended to limit the invention.

[0021] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification controls.

[0022] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the absence of a combination when interpreted in the alternative ("or").

[0023] It is specifically contemplated that the various features of the invention described herein can be used in any combination, unless the context dictates otherwise. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features described herein can be excluded or omitted. For purposes of illustration, if the present specification states that a complex comprises components A, B, and C, it is specifically contemplated that any of A, B, or C, or combinations thereof, can be omitted or discarded.

[0024] As used herein, the transitional phrase "consisting essentially of" (and grammatical variations thereof) should be construed to include materials or steps recited in the claims and that do not materially affect one or more of the basic and novel characteristics of the claimed invention. In re Herz, 537 F.2d 549,551-52,190 USPQ 461,463 (CCPA 1976) (emphasis in original); see also MPEP § 2111.03. Accordingly, the term "consisting essentially of" as used herein should not be construed as equivalent to "comprise."

[0025] As used herein, the words "example," "exemplary," and grammatical variations thereof are intended to refer to non-limiting example 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.

[0026] The term "about," as used herein when referring to a measurable value, e.g., an amount or concentration, is intended to encompass a variation of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified value, as well as the specified value. For example, "about X," where X is a measurable value, is meant to include X and a variation of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of X. Ranges provided herein for measurable values ​​can encompass other ranges and / or individual values ​​therein.

[0027] The recitation of ranges of values ​​herein, unless otherwise stated herein, is intended to serve merely as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated herein as if it were individually set forth herein. For example, if a range of 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed.

[0028] As used herein, a "portion" or "fragment" of a substance or component refers to less than all (e.g., less than 100%) of the substance or component or all of its measurable value. In some embodiments, a "portion" or "fragment" of a substance 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% of the substance or component, or of a measurable value (e.g., a portion of the length, volume, weight, sequence, etc.) of the substance or component. In some embodiments, a "portion" or "fragment" of a particle or particles refers to about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, or about 99%, respectively, of the particle or particles.In some embodiments, the substance or component may be a nucleotide sequence or polypeptide (optionally including a domain), and a "portion" or "fragment" of a nucleotide sequence or polypeptide refers to a nucleotide sequence or polypeptide of a shortened length relative to the reference nucleotide sequence or polypeptide (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more). a shortened length (e.g., one or more nucleotides or one or more peptides) of consecutive residues of the reference nucleotide sequence or polypeptide, respectively, that is 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, and / or consisting essentially of. In some embodiments, a "portion" or "fragment" of a nucleotide sequence or polypeptide has similar or identical biological activity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, or 130% similar biological activity) compared to the reference nucleotide sequence or polypeptide. In some embodiments, a "portion" or "fragment" of a nucleotide sequence or polypeptide has similar or identical biological activity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, or 130% similar biological activity). "SEQ ID NO: 1" has 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% or more, 140% or more, 145% or more, 150% or more, 155% or more, 160% or more, 165% or more, 170% or more, 175% or more, 180% or more, 185% or more, 190% or more, 195% or more, 200% or more increased biological activity) compared to the reference nucleotide sequence or polypeptide.

[0029] As used herein, the words "comprises" and "comprising" specify the presence of stated features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof.

[0030] As used herein, the words "increase," "increase," "enhance," "enhance," "improve," and "enhance" (and grammatical variations thereof) refer to an increase of at least about 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500% or more compared to another measurable characteristic or quantity (e.g., a control value).

[0031] As used herein, the terms "reduction," "reduced," "reduce," "reduce," and "decrease" (as well as grammatical variations thereof) refer to, for example, a decrease of at least about 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 100%, compared to another measurable characteristic or quantity (e.g., a control value). In some embodiments, the decrease may result in no detectable activity or quantity, or essentially no decrease (i.e., an insignificant amount, e.g., less than about 10%, or even less than 5%).

[0032] Particles comprising hyaluronidase are provided according to embodiments of the present invention. In some embodiments, the particles comprising hyaluronidase further comprise an excipient (e.g., a stabilizer) and / or a therapeutic agent. The particles of the present invention may be solid and may 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. In some embodiments, the particles have a water content of less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or less than about 0.5% by weight of the particle. In some embodiments, the particles of the present invention have a moisture content ranging from about 0%, about 0.5%, about 1%, about 2%, about 3%, or about 4% to about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% by weight of the particle. In some embodiments, the particles of the present invention 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 invention have a moisture content of about 0%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% by weight of the particle. In some embodiments, the particles of the present invention have a water activity of less than about 0.9, less than about 0.85, less than about 0.8, less than about 0.75, less than about 0.7, less than about 0.65, less than about 0.6, less than about 0.55, less than about 0.5, less than about 0.45, less than about 0.4, less than about 0.35, less than about 0.3, less than about 0.25, less than about 0.15, less than about 0.1, less than about 0.05, or less than about 0.01. In some embodiments, the particles of the present invention 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 ranging from about 0, about 0.01, about 0.05, about 0.1, about 0.15, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, or about 0.45 to about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, or about 0.9.

[0033] Water content can be measured using methods known in the art, such as Karl Fischer titration and / or thermogravimetric analysis. In some embodiments, the water content of the particles of the present invention can be measured using a Mettler Toledo coulometric KF titrator. A sample containing the particles of the present invention is contacted with a solvent (e.g., methanol) to extract water from the sample, and the amount of water in the solvent is then measured (e.g., using a Mettler Toledo coulometric KF titrator). The amount of water present in the sample can be calculated by subtracting the water present in the solvent blank (the solvent before contact with the particles) from the measured water content of the solvent. For example, in some embodiments, about 15 mg to about 30 mg of particles of the present invention (e.g., in powder form) or a composition (e.g., a suspension) containing particles of the present invention 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 may then be injected into a titration cell (e.g., a Mettler-Toledo coulometric KF titrator), and the amount of water present in the methanol may be measured. The amount of water present in the particles or composition may then be calculated by subtracting the water present in a methanol blank from the measured water content of the methanol. In some embodiments, the water content of a suspension may be measured by directly injecting the suspension into a titration cell. In cases where no volatile components are present in the sample, or where no components are present in the sample that evaporate at a temperature similar to that of water, the water content may also be measured by loss on drying (e.g., using thermogravimetric analysis (TGA)).

[0034] Water activity is a measure of the chemical availability of water in a sample (e.g., in a particle of the present invention) and can quantify the amount of unbound water in the sample and / or how strongly water is bound to components in the sample (e.g., how strongly water is bound to a biological agent present in the particle). In some embodiments, water activity is measured by equilibrating a sample containing a particle of the present invention in a closed chamber and measuring the relative humidity of the headspace in the chamber. Alternatively, or in addition, water activity can be measured by correlating water activity with water content in a known system. To correlate water content with water activity, the water activity of a sample containing a particle of the present invention is determined by equilibrating the sample through a vapor phase with a known water activity (e.g., using a saturated salt solution or a dynamic vapor sorption instrument) and measuring the change in water content by Karl Fischer titration or measuring the change in mass to calculate the water activity of the sample. Thus, the water content and / or mass of a sample is measured before and after contact with the vapor phase, and the change is used to measure the water activity of the sample. In some embodiments, the water content and water activity of a single particle of the invention or a droplet containing a particle of the invention may be correlated by measuring the volume change of the single particle or droplet in response to contact (e.g., exposure to) a solvent with a known water activity or a gas with a known water activity, as described, for example, in Rickard, et al., Biophysical Journal, 2010;98(6):1075-84. The volume of a single particle or droplet is measured before and after contact with the solvent or gas, and the change in volume is used to measure the water activity of the sample.

[0035] In some embodiments, the particles of the present invention have a moisture content (e.g., total moisture content) of less than about 3% of the total particle mass. The particles may have a total moisture content of less than about 2.5%, less than about 2%, less than about 1.5%, less than about 1%, or less than about 0.5% of the total particle mass. In some embodiments, the total moisture content of the particles ranges from about 0%, about 0.1%, or about 0.5% to about 1%, about 1.5%, about 2%, about 2.5%, or about 3% of the total particle mass.

[0036] The particles of the present invention have a residue content of less than about 3% by weight of the particle (e.g., total particle mass). The particles may have a residue content of less than about 2.5%, less than about 2%, less than about 1.5%, less than about 1%, or less than about 0.5% by weight of the particle. In some embodiments, the residue content of the particles of the present invention ranges from about 0%, about 0.01%, about 0.05%, about 0.1%, or about 0.5% to about 1%, about 1.5%, about 2%, about 2.5%, or about 3% by weight of the particle. In some embodiments, the particles of the present invention have a residue content of less than 3% by weight of the particle (e.g., the sum of all residues), for example, less than 2.5%, less than 2%, less than 1.5%, less than 1%, or less than 0.5% by weight of the particle. In some embodiments, a single residue may be present in the particles of the present invention in an amount of less than 1% by weight of the particle, for example, from about 0.01%, about 0.05%, about 0.1%, or about 0.2% to about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, or about 0.9% by weight of the particle. In some embodiments, a single residue may be present in the particles of the present invention in an amount of less than 0.5% by weight of the particle, for example, about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, or about 0.4% by weight of the particle. The residue may be one or more components present in the composition used to prepare (e.g., form, dehydrate, solidify, wash, and / or isolate) the particles of the present invention. Exemplary residues include, but are not limited to, solvents (e.g., octanol and / or pentanol). In some embodiments, the residuals may be Class II or Class III residual solvents as classified by the US Food and Drug Administration.

[0037] The particles of the present invention can be microparticles or nanoparticles. In some embodiments, the particles can have a size in at least one dimension and / or a diameter ranging from about 1 nm, about 5 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, or about 50 nm to about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, or about 500 nm. In some embodiments, the particles of the present invention have an average size of about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, or about 500 nm. In some embodiments, the particles of the present invention may have a size in at least one dimension and / or a diameter ranging from about 0.1 micron, about 0.5 micron, about 1 micron, about 5 micron, about 10 micron, or about 15 micron, to about 20 microns, about 25 microns, or about 30 microns. In some embodiments, the particles may have a size in at least one dimension and / or a diameter from about 0.1 microns, about 0.5 microns, about 1 micron, or about 2 microns to about 3 microns, about 4 microns, about 5 microns, about 6 microns, about 7 microns, about 8 microns, about 9 microns, or about 10 microns. In some embodiments, the particles may have a size in at least one dimension and / or a diameter from about 5 microns, about 6 microns, about 7 microns, about 8 microns, about 9 microns, or about 10 microns to about 11 microns, about 12 microns, about 13 microns, about 14 microns, about 15 microns, about 16 microns, about 17 microns, about 18 microns, about 19 microns, or about 20 microns.In some embodiments, the particles can have a size in at least one dimension and / or diameter of from about 10 microns, about 11 microns, about 12 microns, about 13 microns, about 14 microns, about 15 microns, about 16 microns, about 17 microns, about 18 microns, about 19 microns, or about 20 microns to about 21 microns, about 22 microns, about 23 microns, about 24 microns, about 25 microns, about 26 microns, about 27 microns, about 28 microns, about 29 microns, or about 30 microns.

[0038] The particles of the present invention may be spherical. In some embodiments, the particles may be amorphous. In some embodiments, at least a portion of the particles may be amorphous, and thus the particles may be partially amorphous. For example, all or a portion of the hyaluronidase may be amorphous in the particles of the present invention, but molecules (e.g., salts) within the hyaluronidase matrix may be crystalline in the particles of the present invention. The particles of the present invention may comprise one or more molecules of hyaluronidase, which may be the same or different from each other, and / or a matrix containing one or more molecules of hyaluronidase, which may be the same or different from each other, optionally wherein the hyaluronidase in the particles is amorphous.

[0039] In some embodiments, the particles of the present invention are in the form of a solid, e.g., a solid particle, and / or a powder. In some embodiments, the particles of the present invention are in the form of a solid when they are in the form of a powder (e.g., a dry powder, e.g., a powder in the absence of a liquid in which the powder is dispersed or suspended) and / or when they are present in a vehicle (e.g., a non-aqueous solvent and / or an aqueous solvent). In some embodiments, the particles of the present invention are in the form of a powder. In some embodiments, the particles of the present invention may be microglassified particles, and / or the hyaluronidase may be in the form of a microglassified hyaluronidase that is a particle and / or is contained in a particle. As used herein, "microglassification" refers to a process of removing water (e.g., dehydrating) from a component (e.g., hyaluronidase or a therapeutic agent) present in an aqueous composition (e.g., dissolved or suspended in the aqueous composition), the removal being carried out by contacting the aqueous composition with an organic phase (e.g., a non-aqueous organic phase) to provide a solid containing the component, wherein the solidified component may be referred to herein as a "microglassified" component. The microglassification process may be carried out in any manner that allows for contacting the organic phase with the aqueous composition containing the component, thereby removing water from the component. For example, in some embodiments, the microglassification process may be carried out by using a homogenizer (e.g., an in-line homogenizer) and / or a microfluidic device to contact the organic phase with the aqueous composition containing the component, and / or by spraying droplets of the aqueous composition containing the component into the organic phase, or vice versa, thereby contacting the organic phase with the aqueous composition. In a micro-vitrification process, the organic phase is a liquid organic phase (e.g., a liquid dehydrated composition) and the aqueous composition is a liquid. Thus, a micro-vitrification process involves a liquid-liquid mixture and / or system.The micro-vitrification process may involve contacting at least two different liquids. In some embodiments, the micro-vitrification process does not include a spray-drying step or spray-drying method, and / or does not involve spraying droplets of an aqueous composition containing the component into an organic phase or vice versa. In some embodiments, the micro-vitrification process without a spray-drying step or spray-drying method does not involve spraying droplets of an aqueous composition containing the component into a gas (e.g., a heated gas phase), and / or the micro-vitrification process does not involve a liquid-gas mixture and / or system. The micro-vitrified component is not a precipitate. In some embodiments, the particles of the present invention are not prepared by a method using a phase separation agent. As used herein, the term "phase separating agent" refers to an agent that precipitates a biological product from solution or that causes a liquid-liquid phase separation in which one liquid phase contains the majority of the biological product. Exemplary phase separating agents include, but are not limited to, salts (e.g., those that can be used for "salting out proteins") and polyethylene glycol (PEG). Although phase separation is a nucleation and growth process that can occur during the microvitrification process (e.g., liquid-liquid and / or liquid-solid), the microvitrified component is not formed by phase separation. Instead, microvitrification is a continuous phase transition that does not require nucleation to form the solidified component. The microvitrified component (e.g., microvitrified hyaluronidase) may redissolve in the original composition (e.g., aqueous solution) in which it was present and / or in an aqueous composition (e.g., aqueous buffer).

[0040] In some embodiments, a microvitrified component (e.g., microvitrified hyaluronidase) may be more stable (e.g., extended shelf life, expanded range of acceptable storage temperatures, and / or reduced degradation, optionally reduced degradation after repeated freeze / thaw cycles) than a solution of the component (e.g., a hyaluronidase solution where the hyaluronidase solution is not microvitrified). In some embodiments, microvitrification of a component (e.g., hyaluronidase) optionally improves the stability of the component compared to the stability of the same component in solution that is not microvitrified. In some embodiments, the hyaluronidase-containing particles of the invention have improved stability compared to hyaluronidase in solution when the hyaluronidase in solution is not particulate and not microvitrified, where the hyaluronidase in solution is not particulate and not microvitrified.

[0041] Particles comprising hyaluronidase can be in any form, such as, for example, microparticles, nanoparticles, microspheres, or nanospheres. The particles of the present invention can be amorphous and / or crystalline. In some embodiments, the particles of the present invention comprise amorphous particles. In some embodiments, the particles of the present invention comprise crystalline particles, for example, when a small molecule (e.g., a salt) that can be crystallized is present. The particles of the present invention can be uniform in size or polydisperse. In some embodiments, at least a portion of the particles of the present invention have a size that is within about ±5%, about ±10%, about ±15%, about ±20%, about ±25%, about ±50%, about ±75%, about ±100%, about ±150%, about ±200%, or greater of the average particle size. In some embodiments, the compositions of the present invention comprise discrete particles. The particles of the present invention have a mass of about 0.5 g / cm. 3 , about 0.6g / cm 3 , about 0.7g / cm 3 , about 0.8g / cm 3 , about 0.9g / cm 3 or about 1 g / cm3 From this, it is approximately 1.1 g / cm 3 , about 1.2g / cm 3 , about 1.3g / cm 3 , approximately 1.4 g / cm 3 , about 1.5g / cm 3 , about 1.6g / cm 3 , about 1.7g / cm 3 , about 1.8g / cm 3 , about 1.9g / cm 3 or about 2 g / cm 3 In some embodiments, the particles of the present invention may have a density of up to about 0.5 g / cm 3 , about 0.6g / cm 3 , about 0.7g / cm 3 , about 0.8g / cm 3 , about 0.9g / cm 3 , about 1g / cm 3 , about 1.1g / cm 3 , about 1.2g / cm 3 , about 1.3g / cm 3 , approximately 1.4 g / cm 3 , about 1.5g / cm 3 , about 1.6g / cm 3 , about 1.7g / cm 3 , about 1.8g / cm 3 , about 1.9g / cm 3 , or about 2 g / cm 3 In some embodiments, the particles of the present invention have a density of about 1.1 g / cm 3 ~Approx. 1.5g / cm 3 The density can be measured and / or determined using methods known to those skilled in the art, for example, using a gas pycnometer and / or by sedimentation experiments.

[0042] As used herein, "hyaluronidase" refers to an enzyme or a portion thereof (e.g., a domain thereof) that cleaves glycosidic bonds in glycosaminoglycans, such as, but not limited to, hyaluronic acid. As used herein, "hyaluronidase" refers to all forms of hyaluronidase from any organism (e.g., bacteria and / or mammals, such as humans and / or cows), including naturally occurring (e.g., wild-type hyaluronidase) and recombinant hyaluronidase. In some embodiments, the hyaluronidase present in the particles of the present invention cleaves at least hyaluronic acid, such as human hyaluronic acid, that may optionally be present in the skin and / or tissue of a subject. The hyaluronidase of the present invention may cleave beta 1-4 bonds in glycosaminoglycans (e.g., beta 1-4 bonds in hyaluronic acid). Exemplary hyaluronidases include, but are not limited to, human hyaluronidase (e.g., human Hyal-1, human Hyal-2, human Hyal-3, human Hyal-4, and / or human PH-20 / Spam1), bovine testicular hyaluronidase, and / or ovine hyaluronidase (e.g., ovine PH-20). For example, in some embodiments, the hyaluronidase may be ovine testicular hyaluronidase V (e.g., Millipore Sigma Catalog Number: H6254), optionally having a specific activity of 1,500 units or more per mg of solid. The hyaluronidase present in the particles of the invention may be synthetically obtained (e.g., synthesized in a laboratory) and / or may be naturally obtained and / or derived (e.g., from living or formerly living organisms). In some embodiments, the hyaluronidase may be the same as a hyaluronidase found in nature (i.e., a native hyaluronidase) or may be modified from a hyaluronidase found in nature (e.g., a modified hyaluronidase and / or a recombinant hyaluronidase).For example, the hyaluronidases of the invention may be modified to have a different structure, sequence, charge, chemical modification, and / or length than a naturally occurring hyaluronidase, and / or the hyaluronidase may be a portion of a naturally occurring hyaluronidase (e.g., the active domain of a naturally occurring hyaluronidase). In some embodiments, the hyaluronidases of the invention are similar to commercially available hyaluronidases (TERGASE). 登録商標 (recombinant human hyaluronidase), ALT-BC4 (recombinant human hyaluronidase), ALT-B4 (recombinant human hyaluronidase), MK-5180 (recombinant human hyaluronidase), HYLENEX 登録商標 (recombinant human hyaluronidase; SEQ ID NO: 1), AMPHADASE 登録商標 (bovine testicular hyaluronidase; SEQ ID NO: 2), HYDASE 商標 (bovine testicular hyaluronidase; SEQ ID NO: 2), VITRASE 登録商標(sheep testicular hyaluronidase, which may optionally have the sequence of SEQ ID NO: 3 and / or SEQ ID NO: 4), Vespula vulgaris hyaluronidase, for example, the hyaluronidase having one of the sequences of SEQ ID NO: 21 or 22, Apis mellifera hyaluronidase, for example, the hyaluronidase having the sequence of SEQ ID NO: 23, Dolichovespula maculata hyaluronidase, for example, the hyaluronidase having the sequence of SEQ ID NO: 24, Polistes annularis hyaluronidase, for example, the hyaluronidase having the sequence of SEQ ID NO: 25, and Mus musculus hyaluronidase. musculus hyaluronidase, for example, the hyaluronidase having any one of SEQ ID NOs: 26 to 28 or 39; brown rat (Rattus norvegicus) hyaluronidase, for example, the hyaluronidase having any one of SEQ ID NOs: 31 to 33 or 38; European rabbit (Oryctolagus cuniculus) hyaluronidase, for example, the hyaluronidase having the sequence of SEQ ID NO: 34; Bornean orangutan (Pongo pygmaeus) hyaluronidase, for example, the hyaluronidase having the sequence of SEQ ID NO: 35; cynomolgus monkey (Macaca fascicularis) hyaluronidase, for example, the hyaluronidase having the sequence of SEQ ID NO: 36; guinea pig (Cavia porcellus hyaluronidase, for example, the hyaluronidase having the sequence of SEQ ID NO: 37; Staphylococcus aureus hyaluronidase, for example, the hyaluronidase having the sequence of SEQ ID NO: 40; Staphylococcus pyogenes hyaluronidase, for example, the hyaluronidase having the sequence of SEQ ID NO: 41; Clostridium perfringens hyaluronidase, for example, the hyaluronidase having the sequence of SEQ ID NO: 42;perfringens hyaluronidase, for example, the hyaluronidase having the sequence of SEQ ID NO: 42, and / or the hyaluronidase according to any one of SEQ ID NOs: 5-20 or 43-896), and / or may have activity equivalent to the commercially available hyaluronidase. In some embodiments, the hyaluronidase of the present invention is human hyaluronidase, bovine hyaluronidase (e.g., bovine testicular hyaluronidase), ovine hyaluronidase (e.g., ovine testicular hyaluronidase), yellow banded hornet (Vespula vulgaris) hyaluronidase, European honeybee (Apis mellifera) hyaluronidase, Japanese hornet (Dolichovespula maculata) hyaluronidase, paper wasp (Polistes annularis) hyaluronidase, house mouse (Mus musculus) hyaluronidase, wild boar (Sus scrofa) hyaluronidase, brown rat (Rattus norvegicus hyaluronidase, European rabbit (Oryctolagus cuniculus) hyaluronidase, Bornean orangutan (Pongo pygmaeus) hyaluronidase, cynomolgus monkey (Macaca fascicularis) hyaluronidase, guinea pig (Cavia porcellus) hyaluronidase, Staphylococcus aureus hyaluronidase, Streptococcus pyogenes hyaluronidase, Clostridium perfringens hyaluronidaseperfringens hyaluronidase, and / or any of the recombinant hyaluronidases described above. In some embodiments, the hyaluronidases of the invention have an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to one or more of SEQ ID NOS: 1-896. In some embodiments, the hyaluronidases of the invention comprise the amino acid sequence of one of SEQ ID NOS: 1-896. In some embodiments, the hyaluronidase may be one described in U.S. Pat. No. 8,431,380; U.S. Pat. No. 10,857,213; U.S. Pat. No. 9,284,543; U.S. Pat. No. 9,447,401; U.S. Patent Application Publication No. 2021 / 0155913; U.S. Patent Application Publication No. US2023 / 0250408; and Stern, R. and Jedrzejas, MJ, 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 entireties.

[0043] The hyaluronidase may be present in the particles of the invention in an amount of about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or about 100% by weight of the particle. In some embodiments, the hyaluronidase is present in the particles of the invention in an amount from about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70% to about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or about 100% by weight of the particle. In some embodiments, the hyaluronidase is present in the particles of the invention in an amount of from about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, or about 1% by weight of the particle to about 2%, about 3%, about 4%, or about 5% by weight of the particle. In some embodiments, the hyaluronidase is present in the particles of the invention in an amount of from about 50%, about 55%, about 60%, about 65%, or about 70% by weight of the particle to about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% by weight of the particle.

[0044] The particles of the present invention, and / or the hyaluronidase and / or therapeutic agent present in the particles, may dissolve in the aqueous composition upon and / or after contact with the aqueous composition. In some embodiments, contacting the particles of the present invention with the aqueous composition may be performed ex vivo and / or in vitro. In some embodiments, contacting the particles of the present invention with the aqueous composition may be performed in vivo, for example, during and / or after administration to a subject (e.g., subcutaneous administration) by contacting the particles with bodily fluids. All or a portion of the particles may dissolve in the aqueous composition, and / or all or a portion of the hyaluronidase and / or other components (e.g., therapeutic agents) present in the particles may dissolve in the aqueous composition. In some embodiments, the particles of the invention and / or components thereof (e.g., hyaluronidase and / or therapeutic agent) may dissolve after contact with the aqueous composition in about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, or about 24 hours or more starting at the time of 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, about 34°C, about 35°C, or about 36°C to about 38°C). In some embodiments, the particles of the invention and / or components thereof (e.g., hyaluronidase and / or therapeutic agent) dissolve in the aqueous composition within about 15 seconds, about 30 seconds, or about 45 seconds, or about 1 minute, about 5 minutes, about 15 minutes, about 30 minutes, or about 45 minutes, or about 1 hour, about 1.25 hours, about 1.5 hours, about 1.75 hours, or about 2 hours after contact of the particles with the aqueous composition, optionally at a temperature ranging from 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).

[0045] The particles of the present invention may maintain and / or preserve one or more properties and / or functions 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 compared to a control hyaluronidase (e.g., free hyaluronidase) and / or control therapeutic agent not provided in particle form. In some embodiments, the particles of the present invention may enhance the stability of the hyaluronidase and / or therapeutic agent present in the particles compared to the stability of the hyaluronidase and / or therapeutic agent prior to formation of the particles. The control hyaluronidase may be and / or correspond to the same hyaluronidase used in the particles of the present invention, and the control therapeutic agent may be and / or correspond to the same therapeutic agent used in the particles of the present invention. In some embodiments, the control hyaluronidase is a commercially available hyaluronidase that is not provided in the form of a particle of the present invention, and the control hyaluronidase may be the same type as the hyaluronidase used in the particles of the present invention.

[0046] In some embodiments, after dissolving all or a portion of the particles of the invention and / or their components (e.g., hyaluronidase and / or therapeutic agent) in an aqueous composition, the activity of the component (e.g., hyaluronidase and / or therapeutic agent) is within about ±5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% of the activity of the control component and / or the activity of the component before formation into a particle of the invention. 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., the enzymatic activity of hyaluronidase) can be measured by methods known to those skilled in the art. For example, hyaluronidase activity can be measured using the USP XXII Hyaluronidase Assay, EC 3.2.1.35, HAse and / or USP 29-NF24, page 1057. In some embodiments, hyaluronidase activity and / or changes in hyaluronidase activity can be determined and / or measured by measuring the viscosity of a composition comprising hyaluronidase and / or comparing the viscosity of a composition comprising hyaluronidase after contact with hyaluronic acid for a period of time. For example, in some embodiments, the viscosity of a composition comprising hyaluronidase can be measured and compared to a standard curve prepared using the viscosity of a composition comprising a specific amount of hyaluronidase after two or more different periods of time after contact with hyaluronic acid. In some embodiments, the control component is a control hyaluronidase and / or a control therapeutic agent. In some embodiments, hyaluronidase activity can be measured using a turbidimetric assay, such as that described in Dorfman, A. (1955) Methods in Enzymology, Volume I, 166-173 (1 unit corresponds to 1 NF unit as determined by the method described in USP XXII-NF XVII combined edition, p. 644 (1990)).For example, in some embodiments, the turbidity (e.g., optical density) of a composition comprising hyaluronidase can be measured as the percent transmittance of 600 nm light passing through 1 cm of a hyaluronic acid composition (e.g., a composition (e.g., a solution) comprising about 0.1 mg / ml, about 0.2 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, or about 0.5 mg / ml of hyaluronic acid present (e.g., dissolved in phosphate buffered saline)). In some embodiments, a reaction mixture containing or consisting of 160 mM sodium phosphate, 39 mM sodium chloride, 0.005% (w / v) bovine serum albumin, 0.015% (w / v) hyaluronic acid, 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 in 1:1 HCl:HO (pH 3.75)) and incubated at room temperature for 10 minutes. The percent transmittance at 600 nm is measured and compared to a standard curve. In some embodiments, one unit of hyaluronidase activity corresponds to a change in A600 of 0.330 per minute in a 2.0 mL 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) hyaluronic acid, and 1.5-5 units of hyaluronidase) at pH 5.35 and 37° C. In some embodiments, the activity of the hyaluronidase and / or therapeutic agent after dissolution is compared to the activity of the hyaluronidase and / or therapeutic agent prior to particle formation of the invention. In some embodiments, after the particles of the present invention are dissolved in an aqueous composition, the enzymatic activity of the hyaluronidase present in the particles is within about ±5%, about ±10%, about ±15%, about ±20%, about ±25%, about ±30%, about ±35%, about ±40%, about ±45%, or about ±50% of the enzymatic activity of a control hyaluronidase (e.g., free hyaluronidase).For example, a hyaluronidase having a specific activity of 1,500 units per solid (wherein the solid is a form of hyaluronidase prior to particle formation, e.g., a lyophilized powder) may be included in and / or used to form a particle of the invention (e.g., the particle comprises 1,500 units of hyaluronidase activity per mg of solid, where the solid is a lyophilized powder of hyaluronidase prior to particle formation), and when the hyaluronidase-containing particle is dissolved (e.g., in whole or in part) in an aqueous composition, the dissolved hyaluronidase may have a specific activity of at least about 750 units per mg of solid. "Free hyaluronidase" refers to hyaluronidase that is not included in a particle of the invention.

[0047] In some embodiments, after storing the particles of the invention and then dissolving the particles (e.g., in whole or in part) in an aqueous composition, the activity of a component (e.g., hyaluronidase and / or therapeutic agent) within the particle can be within about ±5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the activity of a control component (e.g., a control hyaluronidase and / or a control therapeutic agent, respectively) and / or of the activity of the component prior to formation of the particle. In some embodiments, after storing particles of the invention and then dissolving the particles (e.g., all or part) in an aqueous composition, the enzymatic activity of the hyaluronidase present in the particles can be within about ±5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the enzymatic activity of a control hyaluronidase and / or of the hyaluronidase prior to formation of the particles.

[0048] In some embodiments, after storing the particles at about 4°C to about 40°C for at least about 3 months, the activity of a component present in the particle (e.g., hyaluronidase and / or therapeutic agent) is within about ±5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% of the activity of a control component and / or the activity of the component prior to the particle of the invention, optionally after dissolving the particle and / or component in an aqueous composition. In some embodiments, the enzymatic activity of the hyaluronidase-containing particles after storage at about 4°C to about 40°C for at least about 3 months is within about ±5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% of the enzymatic activity of a control hyaluronidase and / or the enzymatic activity of the hyaluronidase before formation of the particles.

[0049] The particles of the present invention can be heated at a temperature ranging from about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C or about 20°C to about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, about 50°C, about 51°C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, about 70°C, about 71°C, about 72°C, about The composition may be stored at a temperature of up to about 3°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C for at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or more weeks, months, and / or years. In some embodiments, the particles of the invention are stored at a temperature of about 2°C to about 8°C or about 10°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 13 months, at least about 14 months, at least about 15 months, at least about 16 months, at least about 17 months, at least about 18 months, at least about 19 months, at least about 20 months, at least about 21 months, at least about 22 months, at least about 23 months, or at least about 24 months. In some embodiments, the particles of the invention are stored at a temperature of about 25°C or about 20°C to about 22°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 13 months, at least about 14 months, at least about 15 months, at least about 16 months, at least about 17 months, at least about 18 months, at least about 19 months, at least about 20 months, at least about 21 months, at least about 22 months, at least about 23 months, or at least about 24 months.In some embodiments, the particles of the invention are stored at a temperature of about 40° C. for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 13 months, at least about 14 months, at least about 15 months, at least about 16 months, at least about 17 months, at least about 18 months, at least about 19 months, at least about 20 months, at least about 21 months, at least about 22 months, at least about 23 months, or at least about 24 months.

[0050] A 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 a particle of the present invention and that can provide a therapeutic effect to a subject. In some embodiments, the therapeutic agent may be an agent that can be microvitrified and that can provide a therapeutic effect to a 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 biological agent, 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, Fv, and / or Fc), a nucleotide, and / or a polynucleotide, such as an oligonucleotide, DNA, and / or RNA. Exemplary antibody therapeutic agents include immunoglobulin G (IgG, human IgG), [fam]-trastuzumab deruxtecan, abagovomab, abciximab, adalimumab, ado-trastuzumab emtansine, emtansine, aducanumab, alemtuzumab, alirocumab, amivantamab, odesivimab-ebgn, anifrolumab, ansuvimab, atezolizumab, atoltivimab, avelumab, axatilimab, bamlanivimab, basiliximab, bebtelovimab, bedinvetmab, belantamab mafodotinmafodotin, belimumab, benralizumab, bevacizumab, bezlotoxumab, bimekizumab, blinatumomab, brentuximab vedotin, brodalumab, brolucizumab, burosumab, camrelizumab, canakinumab, caplacizumab, casirivimab, catumaxomab, cemiplimab, certolizumab pegol pegol, cetuximab, silgavimab, concizumab, cosibelimab, crizanlizumab, crovalimab, daclizumab, daratumumab, denosumab, dinutuximab, dinutuximab beta beta), donanemab, dostarlimab, dupilumab, durvalumab, eculizumab, edrecolomab, efalizumab, elotuzumab, elranatamab, emapalumab, emicizumab, enfortumab vedotinvedotin, epcoritamab, eptinezumab, erenumab, ertumaxomab, etesevimab, evinacumab, evolocumab, faricimab, fremanezumab, frunevetmab, galcanezumab, garadacimab, gemtuzumab, gemtuzumab ozogamicin ozogamicin, glofitamab, golimumab, guselkumab, ibalizumab, ibritumomab tiuxetan, idarucizumab, imdevimab, inebilizumab, infliximab, inotuzumab, inotuzumab ozogamicin ozogamicin, ipilimumab, isatuximab, itolizumab, ixekizumab, lanadelumab, lebrikizumab, lecanemab, lokivetmab, loncastuximab tesirine, maftivimab, margetuximab, marstacimab, mepolizumab, mirikizumab, mirvetuximab soravtansine soravtansine, mogamulizumab, mosunetuzumab, moxetumomabpasudotox, muromonab-cd3, narsoplimab, natalizumab, naxitamab, nebacumab, necitumumab, nimotuzumab, nirsevimab, nivolumab, obilturonate, obilturonate Obiltoxaximab, obinutuzumab, ocrelizumab, odronextamab, ofatumumab, olaratumab, omalizumab, palivizumab, panitumumab, patritumab deruxtecan, pembrolizumab, pertuzumab, polatuzumab vedotin Vedotin, pozelimab, racotumomab, ramucirumab, ranibizumab, ravulizumab, raxibacumab, regdanvimab, relatlimab, reslizumab, retifanlimab, risankizumab, rituximab, romosozumab, rozanolixizumab, ruplizumab, sacituzumab govitecangovitecan, sarilumab, satralizumab, secukinumab, serplulimab, siltuximab, sintilimab, sotrovimab, spesolimab, sugemalimab, sutimlimab, tafa Tafasitamab, talquetamab, tarlatamab, tebentafusp, teclistamab, teplizumab, teprotumumab, tezepelumab, tildrakizumab, tislelizumab, tisotumab Examples of anti-cancer drugs include, but are not limited to, vedotin, tixagevimab, tocilizumab, toripalimab, tositumomab, tralokinumab, trastuzumab, tremelimumab, ublituximab, ustekinumab, vedolizumab, veltuzumab, and zolbetuximab, and fragments of any of the foregoing.

[0051] In some embodiments, the therapeutic agent is IgG (human IgG), abagovomab, adalimumab, alirocumab, bedinvetmab, belimumab, benralizumab, bimekizumab, brodalumab, burosumab, canakinumab, casirivimab, certolizumab pegol pegol, concizumab, crovalimab, daclizumab, denosumab, dupilumab, efalizumab, erlanatamab, emicizumab, epcoritamab, erenumab, evolocumab, fremanezumab, frunevetmab, galcanezumab, garadacimab, guselkumab, ixekizumab, lanadelumab, lebrikizumab lebrikizumab, lokivetmab, marstacimab, mepolizumab, mirikizumab, ofatumumab, omalizumab, pozelimab, risankizumab, romosozumab, rozanolixizumab, sarilumab, satralizumab, secukinumab, talquetamab, teclistamab, tezepelumab, tildrakizumab,Tocilizumab, tralokinumab, ustekinumab, veltuzumab, any fragment thereof, or any combination thereof. In some embodiments, the antibody therapeutic agent is IgG (human IgG), abagovomab, alemtuzumab, daratumumab, denosumab, pertuzumab, rituximab, trastuzumab, veltuzumab, etc., any fragment thereof, or any combination thereof.

[0052] In some embodiments, the therapeutic agent is an antibody conjugated (e.g., covalently attached) to another moiety (e.g., a second therapeutic agent), such as, but not limited to, an antibody drug conjugate (ADC) and / or an antibody-oligo conjugate (AOC). In some embodiments, the therapeutic agent comprises an antibody conjugated to a tubulin inhibitor (e.g., an auristatin, a maytansinoid, and / or tubulysin), a DNA damaging agent (e.g., a calicheamicin, duocarmycin, exatecan, deruxtecan, govitecan, and / or a pyrrolobenzodiazepine), and / or an immunomodulator (e.g., a toll-like receptor agonist and / or an interferon gene stimulator agonist).

[0053] In some embodiments, the therapeutic agent is a small molecule having a molecular weight of less than 500 daltons, and 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, antibacterial agents, anti-inflammatory agents, analgesics, anesthetics, antihistamines, antiseptic agents, immunosuppressants, hemostatic agents, vasodilators, wound healing agents, antitumor agents, antacids, antianxiety agents, antiarrhythmic agents, antibacterial agents, antibiotics, anticoagulants, thrombolytic agents, anticonvulsants, antidepressants, antidiarrheals, antiemetics, antifungal agents, antihypertensive agents, antipsychotics, antipyretics, antivirals, barbiturates, beta-blockers, bronchodilators, cold medicines, corticosteroids, antitussives, cytotoxic agents, decongestants, diuretics, expectorants, hormones, hypoglycemic agents, laxatives, muscle relaxants, sedatives, sex hormones (female and / or male), hypnotics, tranquilizers, vitamins, and / or anti-biofilm agents. In some embodiments, the therapeutic agent is an anti-inflammatory agent, such as a corticosteroid. In some embodiments, the therapeutic agent is an anesthetic, such as bupivacaine. In some embodiments, the therapeutic agent is a hormone, such as testosterone (e.g., testosterone enanthate), dihydrotestosterone, androstenedione, estrone, estradiol, estriol, estradiol, esterol, estrogen, and / or progesterone. In some embodiments, the hormonal therapeutic agent is dissolved in a non-aqueous vehicle (e.g., oil, e.g., sesame oil) that may optionally contain the particles of the present invention. In some embodiments, the therapeutic agent is XYOSTED 登録商標 In some embodiments, the compositions of the present invention comprise a non-aqueous vehicle (e.g., an oil, such as sesame oil), a XYOSTED steroid compound, optionally dissolved in the non-aqueous vehicle (e.g., testosterone enanthate). 登録商標and optionally a particle of the invention. One or more (e.g., 1, 2, 3, 4, 5, or more) different therapeutic agents may be present in the particles and / or compositions of the invention. In some embodiments, two or more different therapeutic agents may be present in the same particle or in different, separate particles.

[0054] The therapeutic agent may be present in the particles of the invention in an amount from about 50%, about 55%, about 60%, about 65%, or about 70% to about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% by weight of the particle. In some embodiments, the particles comprise the therapeutic agent in an amount of about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% by weight of the particle. In some embodiments, the therapeutic agent is present in the particle in an amount from about 75% or about 80% to about 85%, about 90%, or about 95% by weight of the particle, for example, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, or about 95% by weight of the particle. In the particles of the present invention, the hyaluronidase and therapeutic agent may not be covalently bound, and / or the excipient (e.g., stabilizer) may not be covalently bound to the therapeutic agent.

[0055] In some embodiments, the particles of the present invention may not include a therapeutic agent.

[0056] Stabilizers may be present in the particles and / or compositions of the present invention. 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., endogenous denatured proteins). In some embodiments, the stabilizer is albumin (e.g., serum albumin and / or recombinant albumin, such as recombinant albumin commercially available from Albumedix Ltd., Nottingham, United Kingdom). In some embodiments, the stabilizer is hyaluronic acid. In some embodiments, the particles of the present invention may contain sugars, such as, but not limited to, sucrose, glucose, trehalose, mannitol, sorbitol, dextrose, maltose, and / or lactose. In some embodiments, the particles of the present invention may 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 particles of the present invention may comprise an albumin, such as, but not limited to, serum albumin. One or more (e.g., 1, 2, 3, 4, or 5) different stabilizers may be present in the particles of the present invention and / or compositions of the present invention. In the particles of the present invention, the hyaluronidase and stabilizer may not be covalently bound, and / or the stabilizer may not be covalently bound to the therapeutic agent.

[0057] The stabilizer may be present in the particles of the present invention in an amount of from about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, or about 20% by weight of the particle to about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% by weight of the particle. In some embodiments, the stabilizer may be present in the particles of the present invention in an amount of about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% by weight of the particle. In some embodiments, a stabilizer (e.g., an amino acid) may be present in a particle of the invention in an amount of from about 0.1%, about 0.5%, about 1%, about 5%, or about 10% by weight of the particle to about 15%, about 20%, about 25%, or about 30% by weight.

[0058] According to some embodiments of the present invention, a composition is provided that includes the particles of the present invention.The composition may include a plurality of particles of the present invention.In some embodiments, the composition is a suspension, and one or more particles, which may 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 solid.

[0059] In some embodiments, the compositions (e.g., suspensions) of the present invention comprise a plurality of particles, and each particle of the plurality of particles comprises hyaluronidase and a therapeutic agent. In some embodiments, the compositions of the present invention comprise a non-aqueous solvent (e.g., a non-aqueous vehicle) and a plurality of particles, wherein each particle of the plurality of particles comprises hyaluronidase and a therapeutic agent. In some embodiments, the compositions of the present invention comprise an aqueous solvent (e.g., an aqueous vehicle) and a plurality of particles, wherein each particle of the plurality of particles comprises hyaluronidase and a therapeutic agent. In some embodiments, the compositions of the present invention comprise a plurality of particles, wherein each particle of the plurality of particles comprises hyaluronidase and a therapeutic agent, and wherein the composition is in the form of a powder.

[0060] In some embodiments, the compositions (e.g., suspensions) of the present invention comprise a plurality of particles, and a first portion of the plurality of particles comprises particles, each particle comprising hyaluronidase and no different therapeutic agent, and a second portion of the plurality of particles comprises particles, each particle comprising hyaluronidase and a different therapeutic agent and no hyaluronidase. In some embodiments, the compositions of the present invention comprise 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 hyaluronidase and no different therapeutic agent), and a second plurality of particles (wherein each particle of the second plurality of particles comprises hyaluronidase and a different therapeutic agent and no hyaluronidase). In some embodiments, the compositions of the present invention comprise an aqueous solvent (e.g., an aqueous vehicle), a first plurality of particles (wherein each particle of the first plurality of particles comprises hyaluronidase and is free of a different therapeutic agent), and a second plurality of particles (wherein each particle of the second plurality of particles comprises hyaluronidase and a different therapeutic agent and is free of hyaluronidase). In some embodiments, the compositions of the present invention comprise a first plurality of particles (wherein each particle of the first plurality of particles comprises hyaluronidase and is free of a different therapeutic agent), and a second plurality of particles (wherein each particle of the second plurality of particles comprises hyaluronidase and a different therapeutic agent and is free of hyaluronidase), wherein the composition is in the form of a powder.

[0061] In some embodiments, the compositions of the present invention comprise a plurality of particles suspended in the composition (i.e., not dissolved in the composition) and one or more components (e.g., therapeutic agents) that are dissolved in the vehicle (e.g., solvent) present in the composition. In some embodiments, one or more components (e.g., therapeutic agents) are dissolved in the non-aqueous vehicle present in the composition. In some embodiments, the one or more components (e.g., therapeutic agents) are dissolved in the aqueous vehicle present in the composition. The one or more components may be different from hyaluronidase. In some embodiments, hyaluronidase is not dissolved in the compositions of the present invention, and / or the compositions of the present invention do not contain dissolved hyaluronidase. In some embodiments, the compositions of the present invention comprise a non-aqueous solvent; a plurality of particles of the present invention (wherein each particle comprises hyaluronidase and is suspended in a non-aqueous solvent); and a therapeutic agent that does not comprise hyaluronidase and is dissolved in the non-aqueous solvent.

[0062] The particles of the present invention may be present in the composition of the present invention as a solid. In some embodiments, the composition may contain particles of the present invention in an amount ranging from about 1 mg, about 5 mg, about 10 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 150 mg, or about 200 mg of particles (e.g., solid) per mL of the composition to about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, or about 700 mg of particles (e.g., solid) per mL of the composition. In some embodiments, the composition is non-aqueous and / or comprises a non-aqueous vehicle. When used herein with reference to a composition or vehicle, "non-aqueous" refers to a composition or vehicle, respectively, that is made from, uses, or is made by a liquid other than water, and has the properties and / or characteristics of a liquid other than water. In some embodiments, a non-aqueous composition or vehicle comprises one or more (e.g., 1, 2, 3, 4, 5, or more) organic liquids that are insoluble in water and / or immiscible or miscible with water. In some embodiments, a composition of the present invention comprises a non-aqueous organic solvent. In some embodiments, a composition and / or vehicle of the present invention comprises water. In some embodiments, a composition and / or vehicle of the present invention is anhydrous. As used herein, "anhydrous" means that water is not added directly to the composition or vehicle when the composition or vehicle is prepared. However, one skilled in the art will recognize that water may be physically and / or chemically absorbed by one or more components in the composition or vehicle (i.e., indirectly added to the composition) at any time during preparation, storage, and / or use of the composition or vehicle. In some embodiments, the term "anhydrous" means that the composition comprises less than 5% by weight of the composition, or any range and / or individual value therein. The compositions of the present invention may have a water content of less than 5%, less than 4.5%, less than 4%, less than 3.5%, less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1%, or less than 0.5% by weight of the composition.Water content may be measured by methods known to those skilled in the art, such as, but not limited to, Karl Fischer titration.

[0063] The compositions of the present invention may comprise a solvent. One or more (e.g., 1, 2, 3, 4, 5, or more) different solvents may be present in the compositions of the present invention. The particles of the present invention may be insoluble in a solvent present in a composition of the present invention, optionally wherein the particles remain soluble for at least about 1 month, at least about 2 months, at least about 3 months at a temperature of about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, or about 20°C to about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C. The particles may remain in the solvent for at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 13 months, at least about 14 months, at least about 15 months, at least about 16 months, at least about 17 months, at least about 18 months, at least about 19 months, at least about 20 months, at least about 21 months, at least about 22 months, at least about 23 months, or at least about 24 months or longer after storage in the composition. In some embodiments, the hyaluronidase and / or therapeutic agent do not dissolve in the solvent and / or compositions of the invention. In some embodiments, the hyaluronidase does not dissolve in the solvent and / or the compositions of the invention and the therapeutic agent dissolve in the solvent and / or compositions of the invention. Thus, the particles and / or hyaluronidase of the invention may remain solid in the compositions of the invention.Exemplary solvents that may be present in the compositions of the present invention include alcohols, such as alcohols having 1 to 20 carbon atoms (i.e., C1-C20 alcohols) or C4-C20 alcohols, alkanes, such as alkanes having 1 to 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 (e.g., benzyl benzoate, fatty acid esters such as ethyl oleate, triglyceride esters such as MIGLYOL. 登録商標 812 (triglyceride esters of saturated caprylic and capric fatty acids derived from coconut oil / palm kernel oil with plant-derived glycerol) and / or MIGLYOL 登録商標Examples of solvents include, but are not limited to, triglyceride esters such as 840 (esters of saturated caprylic and capric fatty acids of plant origin with propylene glycol, also known 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); lactates such as butyl lactate and / or ethyl lactate; and / or fluorinated compounds such as perfluorocarbons (e.g., perfluorodecalin, perfluorooctane, and / or perfluorooctyl bromide) and / or semifluorinated alkanes (e.g., perfluorobutylbutane, perfluorobutylpentane, perfluorobutylhexane, perfluorobutyloctane, perfluorohexylhexane, perfluorohexyloctane, and / or perfluorohexyldodecane). In some embodiments, the solvent may contain carbon atoms, nitrogen atoms, or sulfur atoms. The compositions of the present invention may contain a solvent that is an ester, for example, an alkyl ester or an aryl ester (e.g., benzyl benzoate). In some embodiments, the compositions of the present invention contain a C1-C20 alcohol, an ester-containing compound (e.g., a C1-C20 ester-containing compound, such as ethyl acetate, butyl acetate, triacetin, isobutyl acetate, isopropyl acetate, isopropyl myristate, methyl acetate, propyl acetate, and / or butyl lactate), acetic acid, acetone, anisole, tert-butyl methyl ether, cumene, dimethyl sulfoxide, diethyl ether, ethyl formate, formic acid, an alkane (e.g., heptane, pentane), and / or methyl ethyl ketone. In some embodiments, the compositions of the present invention contain benzyl benzoate, ethyl oleate, a triglyceride ester, ethyl lactate, and / or sesame oil. In some embodiments, the solvent is an aqueous solvent (e.g., water, saline, etc.).

[0064] The compositions of the present invention may have a viscosity of greater than 1 cP (centipoise) when measured at 25 degrees Celsius. In some embodiments, the compositions of the present invention may be suspensions comprising a plurality of particles suspended in one or more (e.g., 1, 2, 3, 4, 5, or more) non-aqueous solutions. In some embodiments, the compositions of the present invention have a viscosity of greater than 1 cP (centipoise) when measured at about 20 degrees Celsius to about 25 degrees Celsius and about 1,000 s -1 ~about 3,000s -1 The viscosity may be from about 20 cP, about 30 cP, about 40 cP, about 50 cP, about 60 cP, or about 70 cP, to about 80 cP, about 90 cP, about 100 cP, about 110 cP, about 120 cP, about 130 cP, about 140 cP, about 150 cP, about 160 cP, about 170 cP, about 180 cP, about 190 cP, or about 200 cP, when measured at a shear rate of

[0065] 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 first particles and second particles, and the first particles comprise a first therapeutic agent, and the second particles comprise a second therapeutic agent different from the first therapeutic agent, optionally wherein hyaluronidase may be present in the first particles and / or the second particles. In some embodiments, the compositions of the invention comprise particles comprising a first therapeutic agent and / or 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.

[0066] 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 invention. Exemplary excipients include, but are not limited to, stabilizers, surfactants (e.g., detergents), tonicity agents, sugars, salts, antimicrobial agents, antioxidants, reducing agents, etc. In some embodiments, the excipient is present in the compositions of the present invention at a concentration of from about 0.01%, about 0.05%, about 0.1%, or about 0.5% to about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 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 (span; 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, e.g., Poloxamer 188), and / or ethoxylated triglycerides (e.g., ethoxylated castor oil).

[0067] 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, tert-butylhydroquinone, and / or vitamin E (e.g., alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol, and / or delta-tocotrienol). Exemplary reducing agents include, but are not limited to, glutathione and / or dithiothreitol (DTT).

[0068] The particles of the present invention can be prepared by removing water (e.g., dissolving and / or extracting) from hyaluronidase present in a composition (e.g., an aqueous composition) that optionally includes a therapeutic agent and / or excipient to provide solid particles. In some embodiments, the particles of the present invention are prepared by contacting an aqueous phase with a solvent phase to dehydrate the hyaluronidase present in one phase and form particles comprising hyaluronidase. The methods of the present invention can provide solid particles comprising hyaluronidase, optionally a therapeutic agent, and optionally an excipient, wherein the solid particles 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. In some embodiments, the methods of the present invention include removing water from hyaluronidase present in the composition and providing solidified hyaluronidase particles (i.e., hyaluronidase in solid form). Once the particles of the present invention are formed, the particles may be separated from any liquid and / or provided in powder form. In some embodiments, the particles of the present invention may be suspended in a solvent (eg, a non-aqueous solvent).

[0069] In some embodiments, the particles of the present invention can be prepared according to a microglassification process. In some embodiments, water can be removed from a composition comprising hyaluronidase using an aqueous phase and a non-aqueous phase to provide a solid comprising hyaluronidase. In some embodiments, the compositions and / or methods used herein (e.g., microglassification methods) do not include a phase separation agent (i.e., there is no phase separation agent). In some embodiments, the particles of the present invention are microvitrified hyaluronidase-containing particles. In some embodiments, the hyaluronidase-containing particles can be redissolved in the initial composition (e.g., aqueous solution), body fluid, and / or aqueous composition (e.g., aqueous buffer) in which they were present. In some embodiments, the method of forming the particles of the present invention can include the apparatus, steps, and / or compositions described in U.S. Pat. No. 8,013,022 and / or U.S. Patent Application Publication No. 2022 / 0119760, which are incorporated herein by reference in their entireties.

[0070] In some embodiments, a composition comprising hyaluronidase (e.g., an aqueous composition) is contacted with a dehydrated composition to prepare the particles of the present invention. As used herein, "contact," "contacting," "contacted," and grammatical variations thereof refer to bringing two or more materials (e.g., compositions, compounds, solvents, etc.) together to form a mixture. Contacting two or more materials may be performed by pouring, spraying, mixing, flowing, injecting (e.g., microinjecting), and / or similar methods, the two materials or portions thereof together. For example, contacting may include adding a solvent to the hyaluronidase-containing composition, or adding the hyaluronidase-containing composition to the solvent. Contacting may be performed in a device, such as a mixer, homogenizer, and / or microfluidic device.

[0071] In some embodiments, contacting involves bringing two or more materials (e.g., compositions, compounds, solvents, etc.) into sufficient proximity that a desired reaction can occur under appropriate 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 have one phase or more (e.g., two, three, or more) phases. In some embodiments, the mixture is a multiphase composition, and the composition has two or more (e.g., two, three, four, 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 containing solid particles). In some embodiments, an emulsion is formed, and the emulsion may be a water-in-oil emulsion and may comprise droplets having a diameter and / or smallest dimension of less than about 1000 μm, less than about 900 μm, less than about 800 μm, less than about 700 μm, less than about 600 μm, less than about 500 μm, less than about 400 μm, less than about 300 μm, less than about 200 μm, less than about 100 μm, less than about 50 μm, less than about 25 μm, less than about 10 μm, less than about 5, or less than about 1 μm. In some embodiments, the mixture is a water-in-oil emulsion comprising droplets having a diameter and / or smallest dimension ranging from about 5 μm, about 10 μm, about 25 μm, about 50 μm, about 100 μm, about 200 μm, about 300 μm, about 400 μm, or about 500 μm to about 600 μm, about 700 μm, about 800 μm, about 900 μm, or about 1000 μm. In some embodiments, the mixture is a water-in-oil emulsion comprising droplets having a diameter and / or smallest dimension ranging from about 0.1 μm, about 0.5 μm, about 1 μm, about 2 μm, about 3 μm, about 4 μm, or about 5 μm to about 6 μm, about 7 μm, about 8 μm, about 9 μm, about 10 μm, about 11 μm, about 12 μm, about 13 μm, about 14 μm, or about 15 μm.In some embodiments, the contacting step forms a mixture (e.g., an emulsion) and a component present in one of the materials (e.g., hyaluronidase) is at least partially dehydrated as a result of the contacting step and / or the step of forming the mixture.

[0072] The particles of the present invention can be formed by contacting a composition comprising hyaluronidase with a dehydrated composition that at least partially dehydrates components present in the composition (e.g., hyaluronidase, a therapeutic agent, and / or excipients present in the composition). Upon contacting the dehydrated composition with a composition comprising hyaluronidase, the dehydrated composition can be present in a concentration and / or volume sufficient to at least partially dehydrate the components of the composition (e.g., hyaluronidase). In some embodiments, the dehydrated composition comprises a solvent (e.g., an organic solvent) that removes water from the components present in the composition (e.g., hyaluronidase and / or a therapeutic agent). Exemplary organic solvents include, but are not limited to, alcohols, e.g., alcohols having 1 to 20 carbon atoms (i.e., C1-C20 alcohols) or C4-C20 alcohols, ester-containing compounds (e.g., C1-C20 ester-containing compounds, e.g., ethyl acetate, butyl acetate, triacetin, isobutyl acetate, isopropyl acetate, methyl acetate, propyl acetate, and / or butyl lactate, etc.), acetic acid, acetone, anisole, tert-butyl methyl ether, cumene, dimethyl sulfoxide, diethyl ether, ethyl formate, formic acid, alkanes (e.g., heptane, pentane, etc.), and / or methyl ethyl ketone, etc.

[0073] In some embodiments, the dehydrated composition comprises a C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, and / or a C20 alcohol, and / or a C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, and / or a C20 ester-containing compound. In some embodiments, the alcohol is selected from methanol, ethanol, propanol (e.g., 1-propanol, 2-propanol); butanol (e.g., 1-butanol; 2-butanol); 2-methyl-1-propanol; 2-methyl-2-propanol; tert-butanol; pentanol (e.g., 1-pentanol, 3-methyl-1-butanol, 2,2-dimethyl-1-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-1-ol; benzyl alcohol; phenylmethanol; diphenylmethanol; undecanol; dodecanol; propyldecanol; butadecanol; pentadecanol; hexadecanol (e.g., 1-hexadecanol); and / or triphenylmethanol. In some embodiments, the dehydrated composition does not include a C10 alcohol (e.g., 1-decanol). In some embodiments, the ester-containing compound can be formed from an acid and a C1-C20, C1-C10, C1-C8, C1-C6, or C1-C4 alcohol.In some embodiments, the dehydrated composition comprises a straight-chain alcohol (e.g., 2-octanol, 3-pentanol, 4-decanol), a derivative of a straight-chain alcohol or its isomer (e.g., octyldodecanol, neopentyl alcohol), a di-, tri-, or quad-hydroxylated material (e.g., 1,4-butanediol, glycerin), an unsaturated alcohol (e.g., a cyclic, olefinic, or alkynyl alcohol, e.g., cyclohexanol, geraniol, oleic alcohol), and / or an alcohol incorporating internal and / or external heteroatoms (e.g., polyethylene glycol, polypropylene glycol, lactate, etc.).

[0074] One or more (e.g., 1, 2, 3, 4, or more) solvents can be present in the dehydrating compositions of the present invention. For example, in some embodiments, at least two alcohols, 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 (e.g., 1-pentanol and 1-hexadecanol). When two or more solvents are included in the dehydrating composition, they can be present in any suitable ratio. In some embodiments, the dehydrated composition comprises two solvents present in the composition in a ratio (by volume or by weight) of about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, or about 1:20.

[0075] The solvent (e.g., organic solvent) present in the dehydrated compositions of the present invention may have a water solubility of from about 0.05 w / w%, about 1 w / w%, about 2 w / w%, or about 5 w / w%, to about 10 w / w%, about 12 w / w%, about 15 w / w%, about 20 w / w%, about 25 w / w%, about 30 w / w%, about 35 w / w%, about 40 w / w%, about 45 w / w%, or about 50 w / w%. In some embodiments, the organic solvent is about 1 w / w%, about 2 w / w%, about 3 w / w%, about 4 w / w%, about 5 w / w%, about 6 w / w%, about 7 w / w%, about 8 w / w%, about 9 w / w%, about 10 w / w%, about 11 w / w%, about 12 w / w%, about 13 w / w%, about 14 w / w%, about 15 w / w%, about 16 w / w%, about 17 w / w%, about 18 w / w%, about 19 w / w%, about 20 w / w%, about 21 w / w%, about 22 w / w%, about 23 w / w%, about 24 w / w%, about 25 w / w%, The solvent (e.g., organic solvent) may have an interfacial tension with water of less than about 55 mN / m. In some embodiments, the solvent present in the dehydrated compositions of the present invention has an interfacial tension with water and / or aqueous compositions that is less than about 55, less than about 50, less than about 45, less than about 40, less than about 35, less than about 30, less than about 25, less than about 20, less than about 15, less than about 10, less than about 9, less than about 8, less than about 7, less than about 6, less than about 5, less than about 4, less than about 3, less than about 2, or less than about 1 mN / m. In some embodiments, the solvent present in the dehydrated compositions of the present invention has an interfacial tension with water and / or aqueous compositions that ranges from about 1, about 2, about 3, about 4, or about 5 to about 6, about 7, about 8, about 9, or about 10.

[0076] In some embodiments, the dehydrated compositions of the present invention comprise one or more (e.g., 1, 2, 3, 4, 5, or more) different organic solvents (e.g., alcohols). In some embodiments, the dehydrated compositions comprise two or more C1-C20 alcohols. In some embodiments, the dehydrated compositions of the present invention comprise about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87 23% by weight, approximately 24% by weight, approximately 25% by weight, approximately 26% by weight, approximately 27% by weight, approximately 28% by weight, approximately 29% by weight, approximately 30% by weight, approximately 31% by weight, approximately 32% by weight, approximately 33% by weight, approximately 34% by weight, approximately 35% by weight, approximately 36 Weight%, about 37% by weight, about 38% by weight, about 39% by weight, about 40% by weight, about 41% by weight, about 42% by weight, about 43% by weight, about 44% by weight, about 45% by weight, about 46% by weight, about 47% by weight, about 48% by weight, about 49% by weight %, about 50% by weight, about 51% by weight, about 52% by weight, about 53% by weight, about 54% by weight, about 55% by weight, about 56% by weight, about 57% by weight, about 58% by weight, about 59% by weight, about 60% by weight, about 61% by weight, about 62% by weight, About 63% by weight, about 64% by weight, about 65% by weight, about 66% by weight, about 67% by weight, about 68% by weight, about 69% by weight, about 70% by weight, about 71% by weight, about 72% by weight, about 73% by weight, about 74% by weight, about 75% by weight, about 7 The organic solvent is present in an amount of about 6%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% by weight.

[0077] The dehydrated composition may include 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 from about 0.01%, about 0.1%, or about 0.5% by weight of the dehydrated composition to about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, or about 35% by weight of the dehydrated composition. In some embodiments, the dehydrated composition includes a surfactant and / or a sugar. In some embodiments, the surfactant may be present in the dehydrated composition in an amount of about 30% by weight or less, such as about 25% by weight or less, about 20% by weight or less, about 15% by weight or less, about 10% by weight or less, about 5% by weight or less, or about 5% by weight or less, etc. In some embodiments, the antimicrobial agent may be present in the dehydrated composition in an amount of about 1% by weight or less, such as about 0.5% by weight or less, about 0.1% by weight or less, or less, etc. In some embodiments, the antioxidant may be present in an amount of about 2.5% by weight or less, such as about 2% by weight or less, about 1.5% by weight or less, about 1% by weight or less, about 0.5% by weight or less, or about 0.1% by weight or less, etc.

[0078] In some embodiments, the dehydration composition comprises at least one alcohol selected from benzyl alcohol, 1-propanol, 1-butanol, tert-butyl ether, sec-butyl ether, 1-pentanol, 1-hexanol, 1-heptanol, 1-octanol, 1-nonanol, 1-decanol, 1-undecanol, 1-dodecanol, propyldecanol, butadecanol, pentadecanol, and hexadecanol, and / or at least one solvent selected from water, triacetin, benzyl alcohol, acetic acid, acetone, anisole, 2-butanol, butyl acetate, tert-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 tert-butanol. Further exemplary solvents that may be present in the dehydrated composition include, but are not limited to, alkanes, such as alkanes having 1 to 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)heteroatom-containing cyclic, acyclic, linear, or branched molecules; and / or lactates. In some embodiments, the dehydrated composition comprises a solvent comprising carbon, nitrogen, and / or sulfur atoms.

[0079] The methods of the invention can include contacting a composition comprising hyaluronidase with one or more (e.g., 1, 2, 3, 4, or more) dehydrated compositions. Thus, the methods can include contacting a composition comprising hyaluronidase with one or more (e.g., 1, 2, 3, 4, or more) solvents, such as a first organic solvent, a second organic solvent, etc. When two or more dehydrated compositions are used, the solvents present in the compositions can be the same or different. In some embodiments, the solvent present in a dehydrated composition can be more volatile than the solvent in the immediately preceding dehydrated composition and / or can be a volatile organic compound (e.g., having a boiling point of from about 0°C, about 50°C, or about 100°C to about 150°C, about 200°C, or about 260°C). In some embodiments, the dehydrated composition can be contacted with the at least partially dehydrated component one or more times (e.g., 1, 2, 3, 4, 5, or more).

[0080] Batch or in-line processes can be used to produce the particles of the present invention by contacting a composition comprising hyaluronidase with one or more dehydrated compositions. In some embodiments, the method comprises contacting a composition comprising hyaluronidase with a first dehydrated composition to form a mixture, and then contacting the mixture with a second dehydrated composition to form the particles of the present invention. In some embodiments, the method comprises contacting a composition comprising hyaluronidase with a first dehydrated 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 dehydrated composition, thereby forming the particles of the present invention.

[0081] The solvent present in the dehydrated composition may form an interface with the composition comprising hyaluronidase. In some embodiments, the solvent present in the dehydrated composition is immiscible with water. In some embodiments, the solvent present in the dehydrated composition first used to dehydrate the component (i.e., the first dehydrated 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 dehydrated composition used after the first dehydrated composition (e.g., the second or third dehydrated composition, or the second or third organic solvent) is immiscible with water.

[0082] Contacting a dehydrated composition with a composition comprising hyaluronidase and / or forming a mixture can reduce the water content of the components (e.g., hyaluronidase and / or therapeutic agent) present in the mixture to at least about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 9 3%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43% 3%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 101%, about 102%, about 103%, about 104%, about 105%, about 106%, about 107%, about 108%, about 109%, about 110%, about 111%, about 112%, about 113%, about 114%, about 115%, about 116%, about 117%, about 118%, about 119%, about 120%, about 121%, about 122%, about 123%, about 124%, about 125%, about 126%, about 127%, about 128%, about 129%, about 130%, about 131%, about 132%, about 133%, about 134%, about 135%, about 136%, about 137%, Including a reduction of 3%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%. In some embodiments, the moisture content of the component is reduced from about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35% to about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% after the contacting step of the present invention.

[0083] A mixture comprising a dehydrated composition and an ingredient (e.g., hyaluronidase and / or a therapeutic agent) can have a water activity of less than about 0.99, less than about 0.98, less than about 0.95, less than about 0.9, less than about 0.85, less than about 0.8, less than about 0.75, less than about 0.7, less than about 0.65, less than about 0.6, less than about 0.55, less than about 0.5, less than about 0.45, less than about 0.4, less than about 0.35, less than about 0.3, less than about 0.25, less than about 0.2, less than about 0.15, or less than about 0.1. In some embodiments, the mixture has a water activity ranging from about 0.1, about 0.15, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, or about 0.5 to about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, or about 0.95. In some embodiments, the mixture has a water saturation of less than about 0.95, less than about 0.9, less than about 0.85, less than about 0.8, less than about 0.75, less than about 0.7, less than about 0.65, less than about 0.6, less than about 0.55, less than about 0.5, less than about 0.45, less than about 0.45, less than about 0.35, less than about 0.3, less than about 0.25, less than about 0.2, less than about 0.15, or less than about 0.1. In some embodiments, the mixture has a water saturation ranging from about 0.1, about 0.15, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, or about 0.5 to about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, or about 0.95. For example, in some embodiments, a mixture containing protein may have a water saturation of less than about 0.95. In some embodiments, a mixture containing salt may have a water saturation of less than about 0.5.

[0084] The at least partially dehydrated components (e.g., hyaluronidase and / or therapeutic agent) can have a water content of about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 1%, or about 0% after contact with one or more dehydration compositions. In some embodiments, the at least partially dehydrated component (e.g., hyaluronidase) has a water content ranging from about 0%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, or about 25% to about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% after contact with one or more dehydration compositions. In some embodiments, the at least partially dehydrated component (e.g., hyaluronidase) has a water content ranging from about 0%, about 0.5%, about 1%, about 2%, about 3%, or about 4% to about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% after contact with one or more dehydration compositions. In some embodiments, the at least partially dehydrated component (e.g., hyaluronidase) has a water activity of about 0.986, about 0.98, about 0.95, about 0.9, about 0.85, about 0.8, about 0.75, about 0.7, about 0.65, about 0.6, about 0.55, about 0.5, about 0.45, about 0.4, about 0.35, about 0.3, about 0.25, about 0.2, about 0.15, about 0.1, about 0.05, or about 0 after contact with one or more dehydration compositions. In some embodiments, the at least partially dehydrated component (e.g., hyaluronidase) has a water activity of about 0, about 0.05, about 0.1, about 0.15, about 0.2, about 0.25, or about 0.3 to about 0.35, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, or about 0.9 after contact with one or more dehydration 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 one or more dehydration 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 one or more dehydration compositions.

[0085] In some embodiments, after contacting the initial dehydrated composition with a composition comprising hyaluronidase, the at least partially dehydrated component (e.g., hyaluronidase) has a water content of less than about 60%, and / or has a water content ranging from about 1%, about 5%, about 10%, about 15%, about 20%, or about 25%, to about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70%. In some embodiments, after contacting the at least partially dehydrated component with a subsequent (e.g., second) dehydrated composition, which may be the same as or different from the initial dehydrated composition, the at least partially dehydrated component has a moisture content of less than about 45% and / or a moisture content of from about 0.5%, about 1%, about 5%, about 10%, about 15%, or about 20%, to about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70%. In some embodiments, the at least partially dehydrated component is a particle of the present invention.

[0086] The contacting step can be carried out at a temperature ranging from about −50° C., about −40° C., about −30° C., about −20° C., about −10° C., about 0° C., about 4° C., about 10° C., about 15° C., or about 20° C. to about 25° C., about 30° C., about 35° C., about 40° C., about 45° C., or about 50° C. In some embodiments, the contacting step in the methods of the invention is carried out at room temperature and / or atmospheric pressure.

[0087] In some embodiments, contacting a dehydrated composition (e.g., an organic solvent) with a composition comprising hyaluronidase and / or forming a mixture comprising the dehydrated composition and a composition comprising hyaluronidase can form a suspension. In some embodiments, the suspension comprises hyaluronidase in the form of a solid suspended in a liquid (e.g., a non-aqueous organic layer). Contacting the dehydrated composition with the composition comprising hyaluronidase to provide a mixture and forming an 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" refers to at least partially dehydrating the component immediately after contact with the dehydrated composition or within less than about 1 minute of initial contact with the dehydrated composition. In some embodiments, forming at least partially dehydrated components in the mixture is achieved within about 100 minutes (e.g., within about 90 minutes, within about 60 minutes, within about 30 minutes, within about 15 minutes, within about 10 minutes, within about 5 minutes, or within about 1 minute) after initial contact of the dehydrated composition with a composition comprising hyaluronidase.

[0088] In some embodiments, an at least partially dehydrated component (e.g., hyaluronidase) having a water content of less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% can be achieved within less than about 10 minutes, less than about 9 minutes, less than about 8 minutes, less than about 7 minutes, less than about 6 minutes, less than about 5 minutes, less than about 4 minutes, less than about 3 minutes, less than about 2 minutes, or less than about 1 minute, or within less than about 50 seconds, less than about 40 seconds, less than about 30 seconds, less than about 20 seconds, less than about 10 seconds, less than about 5 seconds, less than about 1 second, less than about 0.5 seconds, or less than about 0.1 seconds. In some embodiments, an at least partially dehydrated component (e.g., hyaluronidase) having a water content of less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% can be achieved within about 0.01 seconds, within about 0.05 seconds, within about 0.1 seconds, or within about 0.5 seconds, to within about 1 second, within about 2 seconds, within about 3 seconds, within about 4 seconds, within about 5 seconds, within about 6 seconds, within about 7 seconds, within about 8 seconds, within about 9 seconds, or within about 10 seconds.

[0089] According to some embodiments, contacting two different compositions (e.g., a dehydrated composition and a composition comprising hyaluronidase, or a composition comprising a solvent and at least partially dehydrated components) includes mixing them together. Mixing can be accomplished by any method known to those skilled in the art, such as vortexing, stirring, static mixing, homogenizing, extruding, pumping, injecting (e.g., microinjecting), adding one composition to the other, spraying one composition into the other, and / or spraying both 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 the other using slow addition, fast addition, or total addition, and mixing can be achieved at low and / or high shear (e.g., about 1-10,000,000 / sec). When two different compositions include an aqueous composition and a hydrophobic composition (e.g., a composition containing an organic solvent), the aqueous composition may 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, impellers, pipeline mixers (turbulent and laminar), static mixers, in-line mixers, and / or microfluidic devices (e.g., microfluidic devices equipped with a cross junction). In some embodiments, the mixer is in-line, such as an in-line homogenizer.In some embodiments, the mixer is a device (e.g., a microfluidic device), and two different compositions may be added or injected into the device, thereby mixing the two different compositions with each other and forming the mixture. Forming the at least partially dehydrated component can include mixing (e.g., homogenizing) two different compositions and / or mixtures.

[0090] In some embodiments, two different compositions forming a mixture and / or a composition comprising at least partially dehydrated components can be mixed in a ratio that is the volume ratio of the two different compositions, for example, in a batch process. For example, for a first composition and a second composition that are contacted to form a mixture in a batch process, the volume ratio can be the ratio of 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 a composition comprising at least partially dehydrated components can be mixed in a ratio based on the feed rates of the two different compositions, for example, in an in-line process. For example, for a first composition and a second composition that are contacted to form a mixture in an in-line process, the ratio can be the ratio of 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.

[0091] In some embodiments, the two different compositions forming the mixture and / or composition comprising at least partially dehydrated components are mixed at a rate of about 1 mL / min, about 5 mL / min, about 10 mL / min, about 25 mL / min, about 50 mL / min, about 75 mL / min, or about 100 mL / min to about 150 mL / min, about 250 mL / min, about 500 mL / min, about 1,000 mL / min, about 1,500 mL / min, about 2,000 mL / min, about 3,000 mL / min, about 4,000 mL / min, about 5,000 mL / min, about 6,000 mL / min, The mixture may be mixed (e.g., homogenized) at a feed rate (e.g., flow rate) ranging from about 7,000 mL / min to about 8,000 mL / min, or from about 5 mL / min, about 10 mL / min, about 25 mL / min, about 50 mL / min, about 75 mL / min, or about 100 L / min to about 150 mL / min, about 250 mL / min, about 500 mL / min, about 750 mL / min, about 1000 mL / min, about 1,500 mL / min, about 2,000 mL / min, about 3,000 mL / min, about 4,000 mL / min, or about 5,000 L / min. In some embodiments, the mixture and / or the two different compositions forming the composition comprising the at least partially dehydrated components are dehydrated at a rate of about 1 L / hr, about 5 L / hr, about 10 L / hr, about 25 L / hr, about 50 L / hr, about 75 L / hr, or about 100 L / hr to about 150 L / hr, about 250 L / hr, about 500 L / hr, about 1,000 L / hr, about 1,500 L / hr, about 2,000 L / hr, about 2,500 L / hr, about 3,000 L / hr, about The components may be mixed (e.g., homogenized) at feed rates (e.g., flow rates) ranging from 3,500 L / hr or about 4,000 L / hr, or from about 10,000 L / hr, about 15,000 L / hr, about 20,000 L / hr, about 25,000 L / hr, or about 30,000 L / hr, to about 50,000 L / hr, about 75,000 L / hr, about 100,000 L / hr, about 125,000 L / hr, or about 150,000 L / hr. The feed rates for each component may be the same or different.

[0092] In some embodiments, two different compositions may be contacted and / or combined in a given volumetric ratio to form a mixture and / or a composition comprising at least partially dehydrated components. The two different compositions may include an aqueous composition (e.g., a composition comprising hyaluronidase and / or a composition comprising water) that provides an aqueous phase, and a solvent-containing composition (e.g., a hydrophobic composition) that provides a solvent phase. In some embodiments, the two different compositions are at about 0.1:100, about 0.2:100, about 0.3:100, about 0.4:100, about 0.5:100, about 0.6:100, about 0.7:100, about 0.8:100, about 0.9:100, about 1:100, about 1.5:100, about 2:100, about 2.5:100, about 3:100, about 3.5:100, about 4:100, about 4.5:100, about 5:100, about 5.5:100, about 6:100, about 6.5:100, about 7:100, about The components may be contacted and / or combined in a feed volume ratio of about 7.5:100, about 8:100, about 8.5:100, about 9:100, about 9.5:100, about 10:100, about 11:100, about 12:100, about 13:100, about 14:100, about 15:100, about 16:100, about 17:100, about 18:100, about 19:100, about 20:100, about 21:100, about 22:100, about 23:100, about 24:100, or about 25:100 (aqueous composition:solvent containing composition). In some embodiments, the two different compositions that form the mixture and / or composition comprising at least partially dehydrated components may be contacted and / or combined in a feed volume ratio ranging from about 0.1:100, about 0.3:100, about 0.5:100, about 0.7:100, or about 1:100, to about 2:100, about 4:100, about 6:100, about 8:100, about 10:100, about 12:100, about 14:100, about 16:100, about 18:100, about 20:100, about 22:100, or about 25:100.

[0093] The at least partially dehydrated components and / or particles of the present invention can be separated from the mixture and / or liquid phase using methods known to those skilled in the art. For example, the at least partially dehydrated components and / or particles of the present invention can be separated from the liquid phase by filtration (e.g., pressure filtration, tangential flow filtration, rotary filtration, centrifugal filtration, disc stack filtration, etc.), cyclone separation, sedimentation (e.g., acoustic resonant field induced precipitation), evaporation, centrifugation, fluidized bed drying, evaporative drying, thermal drying, freeze drying, atmospheric freeze drying, spray drying, spray freeze drying, microwave / infrared drying, and / or sieving. In some embodiments, the at least partially dehydrated components and / or particles of the present invention can be separated from the liquid phase by washing and / or solvent exchange methods, precipitation and / or filtration processes, and further dried (e.g., to remove water and / or solvent) by air, vacuum, and / or freeze-drying processes, and / or any combination thereof. In some embodiments, the at least partially dehydrated components and / or particles of the present invention can be separated from the liquid phase by washing and / or solvent exchange methods, precipitation and / or filtration processes, and / or further dried (e.g., to remove water and / or solvent) using air, vacuum, and / or freeze-drying processes, and / or any combination thereof.

[0094] In some embodiments, the method of the present invention includes washing the at least partially dehydrated components and / or particles of the present invention with a cleaning composition. The cleaning composition may contain an organic solvent, such as an alcohol (e.g., a C1-C20 alcohol), an alkane (e.g., a C4-C20 alkane), an ester and / or an ether, etc. Such organic solvents include, but are not limited to, those described above, and / or the organic solvent may have water-solubility, as described above. In some embodiments, the organic solvent in the cleaning composition is miscible with water. The organic solvent in the cleaning composition may be the same as or different from the organic solvent in the dehydrated composition. In some embodiments, the organic solvent present in the cleaning composition may be a Class II or Class III residual solvent as classified by the U.S. Food and Drug Administration.

[0095] In some embodiments, the organic solvent present in the cleaning composition may be more volatile than the organic solvent in the dehydration composition. In some embodiments, the organic solvent in the cleaning composition is a volatile organic compound (e.g., having a boiling point of from about 0°C, about 50°C, or about 100°C to about 150°C, about 200°C, or about 260°C). In some embodiments, the cleaning composition can be used to rinse and / or remove the dehydration composition, organic solvent, and / or impurities. In some embodiments, the organic solvent present in the cleaning composition has solubility in the solvent present in the dehydration composition.

[0096] According to some embodiments, the method of the present invention is a one-step method in which a composition containing hyaluronidase is contacted with a dehydration composition to provide the mixture and particles of the present invention. In the one-step method, the contacting step can be repeated one or more times (e.g., 1, 2, 3, 4, 5 or more times) using the same or different dehydration compositions. The one-step method can further include contacting the at least partially dehydrated components or a composition containing the same components with a cleaning composition, which can optionally include an organic solvent that is more volatile than one or more previous organic solvents used in the method.

[0097] The methods of the present invention may involve contacting the dehydrating or cleaning composition with a composition comprising hyaluronidase, at least partially dehydrated components, or a composition comprising at least partially dehydrated components for a period of time, where the contact time for each of these steps may be about 1 minute, about 5 minutes, about 15 minutes, about 30 minutes, about 45 minutes, or about 60 minutes, or about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours, or more. In some embodiments, the contact time is from about 1 minute, about 5 minutes, or about 15 minutes to about 30 minutes, about 45 minutes, or about 60 minutes, or from about 1 hour, about 2 hours, about 3 hours, about 4 hours, or about 5 hours to about 6 hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hours, or more.

[0098] In some embodiments, a composition comprising at least partially dehydrated components (e.g., particles of the invention) has a pH greater than 7, e.g., about 7.5, about 8, about 8.5, about 9, about 10, etc., or higher. In some embodiments, a composition comprising at least partially dehydrated components has a pH less than 7, e.g., about 6.5, about 6, about 5.5, about 5, about 4.5, about 4, or lower. In some embodiments, a composition comprising at least partially dehydrated components has a pH at least ±0.5, at least ±1, at least ±1.5, or at least ±2 pH units away from the isoelectric point (pI) of the desired separated component (e.g., hyaluronidase). In some embodiments, the pH of a composition comprising at least partially dehydrated components in a method of the invention varies by less than about ±2, less than about ±1.5, less than about ±1, or less than about ±0.5 pH units from the initial pH of the composition.

[0099] Some embodiments of the present invention provide a composition comprising a plurality of solid particles (e.g., microparticles, nanoparticles) described herein. In some embodiments, the composition is non-aqueous. The plurality of solid particles included 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 may comprise crystalline particles, for example, in the presence of small molecules (e.g., salts) that can be crystallized. In some embodiments, the composition is provided and / or obtained after separating the solid particles from a liquid phase. The plurality of solid particles may be uniform in size or polydisperse. In some embodiments, at least some of the plurality of solid particles have a size that is within about ±5%, about 10%, about 15%, about 20%, about 25%, about 50%, about 75%, about 100%, about 150%, about 200%, or more of the average particle size. In some embodiments, the composition comprises discrete particles. In some embodiments, the composition comprises a mass of the biological agent (e.g., hyaluronidase and / or protein) in an amount less than about 50%, less than about 40%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than 1% by weight of the biological material.

[0100] In some embodiments, the particles of the present invention have a size configured to pass through pores formed in the hyaluronic acid network when hyaluronidase (e.g., hyaluronidase present in the particles of the present invention and / or hyaluronidase from the particles of the present invention) degrades the hyaluronic acid network, and / or a size that allows the particles of the present invention to pass through pores in the hyaluronic acid network, thereby allowing the particles of the present invention to migrate away from the site of administration to a subject (e.g., an injection site). In some embodiments, the particles of the present invention 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, a plurality of particles of the present invention 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 present invention travel and / or disperse an increased distance from the administration site compared to the same compound or compounds present in the particles when not administered in the form of particles of the present invention. In some embodiments, the particles of the present invention do not disperse from the administration site (e.g., injection site) and / or remain localized at the administration site. When the administered particles of the present invention dissolve, the components of the particles (e.g., hyaluronidase and / or therapeutic agent) may disperse from the administration site. In some embodiments, the particles of the present invention have a size of about 300 nm or greater, and the particles do not disperse from the administration site (e.g., injection site) and / or remain localized at the administration site.

[0101] In some embodiments, at least some of the solid particles in the compositions of the invention are solid particles of the invention comprising solidified hyaluronidase. In some embodiments, at least some of the solid particles in the compositions of the invention are solid particles comprising a solidified therapeutic agent, optionally having 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, at least some of the solid particles in the compositions of the invention comprise both solidified hyaluronidase and a solidified therapeutic agent, either contained within the same particle or within different particles. In some embodiments, a composition comprising a plurality of solid particles comprises at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 100% by weight of the composition as solidified hyaluronidase particles. In some embodiments, the solid particles comprising solidified hyaluronidase can be present in the composition in an amount from about 1 mg / mL, about 5 mg / mL, about 10 mg / mL, about 25 mg / mL, about 50 mg / mL, about 75 mg / mL, about 100 mg / mL, about 150 mg / mL, or about 200 mg / mL to about 300 mg / mL, about 400 mg / mL, about 500 mg / mL, about 600 mg / mL, or about 700 mg / mL.In some embodiments, a composition comprising a plurality of solid particles comprises at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 100% by weight of the composition. In some embodiments, the plurality of solid particles has a moisture content of less than about 15% by weight of the particles and / or a water activity of less than about 0.9. In some embodiments, the plurality of solid particles has a moisture content of less than about 10% by weight of the particles and / or a water activity of less than about 0.5.

[0102] The present invention provides a method for increasing the dissolution of a therapeutic agent in a liquid, the method comprising combining the liquid, the therapeutic agent, and particles comprising a hyaluronidase of the present invention. In some embodiments, the particles comprising the hyaluronidase dissolve in the liquid. In some embodiments, the liquid is an aqueous liquid (e.g., a body fluid and / or an aqueous buffer). The therapeutic agent may be present in the particles comprising the hyaluronidase, or may be separate from the particles comprising the hyaluronidase. In some embodiments, the therapeutic agent may be in particles different from the particles comprising the hyaluronidase, and the particles comprising the therapeutic agent may 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, and may be solid particles. In some embodiments, the therapeutic agent may be separate from the particles comprising the hyaluronidase, and may be present in the composition in which the particles are present (e.g., dissolved and / or suspended in the composition). In some embodiments, the particles comprising hyaluronidase are present in a solvent different from the liquid. In some embodiments, the therapeutic agent is present in a non-aqueous composition and the particles are present in a non-aqueous composition, optionally wherein the therapeutic agent and particles 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 particles are present in a non-aqueous composition.

[0103] In some embodiments, a method for increasing dissolution of a therapeutic agent in a liquid (e.g., in vitro and / or in vivo) can include separately combining particles comprising a therapeutic agent and hyaluronidase with the liquid. For example, the therapeutic agent and particles comprising hyaluronidase can be combined (e.g., added) sequentially with the liquid. In some embodiments, the combining includes simultaneously combining particles comprising hyaluronidase with 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 when combining the liquid with the particles comprising hyaluronidase, the composition comprising the liquid and / or particles can be free of hyaluronidase in solution (e.g., hyaluronidase dissolved in water) and / or free hyaluronidase. When the liquid and the particles containing the hyaluronidase are combined, the hyaluronidase and / or particles can dissolve in the liquid.

[0104] The methods of the invention may increase the solubility of a therapeutic agent in a liquid by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or more over a period of time (e.g., from about 1 minute to about 2 hours) compared to dissolution of the therapeutic agent in the same liquid over the same predetermined period of time and under the same conditions (e.g., temperature and / or pressure) in the absence of the particles of the invention. In some embodiments, after combining the hyaluronidase-containing particles of the invention, the therapeutic agent, and a liquid, at least about 90%, at least about 95%, at least about 99%, or at least about 100% of the therapeutic agent is dissolved within about 10, 20, 30, or 40 minutes to about 50, 60, 70, 80, 90, 100, 110, or 120 minutes. In some embodiments, after initially combining the hyaluronidase-containing particles of the invention, the therapeutic agent, and a liquid, at least about 90%, at least about 95%, at least about 99%, or at least about 100% of the therapeutic agent is dissolved within about 10 minutes, about 20 minutes, about 30 minutes, or about 40 minutes, to about 50 minutes, or about 60 minutes.

[0105] Some embodiments of the present invention provide methods for improving tolerance and / or increasing absorption of a therapeutic agent by administering it to a subject, the method comprising administering a hyaluronidase-containing particle of the present invention to the subject. In some embodiments, the method comprises administering the therapeutic agent and / or particles comprising the hyaluronidase to the subject subcutaneously, intramuscularly, and / or intradermally. In some embodiments, the therapeutic agent and / or particles can be administered to the subject via subcutaneous administration, intramuscular administration, intradermal administration, etc., using methods and / or devices known to those skilled in the art. Exemplary devices include, but are not limited to, syringes (e.g., pre-filled syringes with a staked needle or a removable needle), dual-chamber syringes, plastic syringes that are filled from a vial by the subject or a caregiver, cartridges, and / or vials. In some embodiments, the syringes or cartridges may be used in combination with an auto injector or pen device. Exemplary needle gauges for 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 agents and / or particles of the invention are injected subcutaneously into a subject. In some embodiments, the methods reduce irritation at the site of administration to the subject (e.g., at the injection site).

[0106] In some embodiments, the therapeutic agent and the particles comprising hyaluronidase are administered to a subject separately, optionally simultaneously or at different times (e.g., sequentially). In some embodiments, the therapeutic agent is administered to a subject first, and then the particles comprising hyaluronidase are administered to the subject. In some embodiments, the particles comprising hyaluronidase are administered to a subject first, and then the therapeutic agent is administered to the subject. In some embodiments, when the therapeutic agent and the particles comprising hyaluronidase are administered separately, the period between the two administrations may be less than about 24 hours, for example, less than about 20 hours, less than about 15 hours, less than about 12 hours, less than about 8 hours, less than about 6 hours, less than about 4 hours, less than about 2 hours, or less than about 1 hour, or less than about 45 minutes, less than about 30 minutes, less than about 15 minutes, less than about 10 minutes, less than about 5 minutes, or less than about 2 minutes. In some embodiments, the therapeutic agent and the particles comprising hyaluronidase are administered to a subject simultaneously. In some embodiments, the therapeutic agent and the particles comprising hyaluronidase are present in the same composition administered to a subject. In some embodiments, the therapeutic agent is present in a non-aqueous composition, and the particles are present in a non-aqueous composition, optionally, wherein the therapeutic agent and the particles may be present in the same composition or in different compositions. In some embodiments, the therapeutic agent and the particles comprising hyaluronidase are present in the same particles administered to a subject, or in separate particles administered to a subject. In some embodiments, the therapeutic agent and the particles comprising hyaluronidase are both administered subcutaneously to a subject. In some embodiments, the therapeutic agent and the particles comprising hyaluronidase are both administered intradermally to a subject. In some embodiments, the therapeutic agent is administered subcutaneously to a subject, and the particles comprising hyaluronidase are administered intradermally to a subject. In some embodiments, the therapeutic agent is administered to the subject intradermally and the particles comprising hyaluronidase are administered to the subject subcutaneously, hi some embodiments, the particles comprising the therapeutic agent and / or hyaluronidase are administered to the subject intramuscularly.

[0107] The particles and / or therapeutic agents of the present invention may be administered to a subject in a volume of from about 0.1 mL, about 0.2 mL, about 0.3 mL, about 0.4 mL, or about 0.5 mL to about 0.75 mL, about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 15 mL, about 20 mL, about 25 mL, about 30 mL, about 35 mL, about 40 mL, about 45 mL, or about 50 mL. In some embodiments, a composition comprising particles of the present invention and / or a therapeutic agent is administered to a subject, wherein the composition has a volume of from about 0.1 mL, about 0.2 mL, about 0.3 mL, about 0.4 mL, or about 0.5 mL to about 0.75 mL, about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 15 mL, about 20 mL, about 25 mL, about 30 mL, about 35 mL, about 40 mL, about 45 mL, or about 50 mL. In some embodiments, the compositions of the invention administered to a subject have a volume of about 0.1 mL, about 0.2 mL, about 0.3 mL, about 0.4 mL, about 0.5 mL, about 0.6 mL, about 0.7 mL, about 0.8 mL, about 0.9 mL, about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 15 mL, about 20 mL, about 25 mL, about 30 mL, about 35 mL, about 40 mL, about 45 mL, or about 50 mL. In some embodiments, the particles and therapeutic agent of the invention are administered to a subject in a total administered 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, e.g., less than about 9 mL, less than about 8 mL, less than about 7 mL, less than about 6 mL, less than about 5 mL, less than about 4 mL, less than about 3 mL, less than about 2 mL, or less than about 1 mL. In some embodiments, the particles and therapeutic agent of the present invention are administered to a subject in a total volume of less than about 2 mL.

[0108] The methods of the present invention may not require 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 invention are in solid form when administered to a subject (e.g., subcutaneously and / or intradermally). In some embodiments, the hyaluronidase contained in the particles and / or the particles dissolve upon administration to the subject, for example, upon contact with the subject's body fluids. Hyaluronidase may be administered to a subject in an amount of from about 0.1%, about 0.5%, or about 1% by weight of the particles to about 2%, about 3%, about 4%, or about 5% by weight. In some embodiments, the methods of the invention may include administering to a subject a composition comprising particles comprising hyaluronidase, and the hyaluronidase may be administered to the subject in an amount of from about 0.05%, about 0.1%, about 0.5%, or about 1% by weight of the composition to about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% by weight of the composition, and / or may be present in the composition in an amount of from about 0.05%, about 0.1%, about 0.5%, or about 1% by weight to about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% by weight of the composition.In some embodiments, the hyaluronidase has a concentration of about 100 activity units to about 50,000 activity units (e.g., about 100 activity units, about 200 activity units, about 300 activity units, about 400 activity units, about 500 activity units, about 600 activity units, about 700 activity units, about 800 activity units, about 900 activity units, about 1,000 activity units, about 1,250 activity units, about 1,500 activity units, about 1,750 activity units, about 2,000 activity units, about 2,250 activity units, about 2,500 activity units, about 2,750 activity units, about 3,000 activity units, about 3,250 activity units, about 3,500 activity units, about 4,000 activity units, about 4,500 activity units, about 5,000 activity units, about 6,000 activity units, about 7,000 activity units, about 8,000 activity units, about 9,000 activity units, about 10,000 activity units, about 11,000 activity units, about 12,500 activity units, about 15,000 activity units, about 16,000 activity units, about 17,000 activity units, about 18,000 activity units, about 19,000 activity units, about 21,000 activity units, about 22,500 activity units, about 25,000 activity units, about 26,000 activity units, about 27,000 activity units, about 28,000 activity units, about 29,000 activity units, about 30,000 activity units, about 31,000 activity units, about 32,000 activity units, about 33,000 activity units, about 34,000 activity units, The compound may be administered to a subject in an amount of about 1,500 active units, about 3,750 active units, about 4,000 active units, about 4,250 active units, about 4,500 active units, about 4,750 active units, about 5,000 active units, about 6,000 active units, about 7,000 active units, about 8,000 active units, about 9,000 active units, about 10,000 active units, about 15,000 active units, about 20,000 active units, about 25,000 active units, about 30,000 active units, about 35,000 active units, about 40,000 active units, about 45,000 active units, or about 50,000 active units. In some embodiments, the hyaluronidase activity in the compositions ranges from about 1 Unit, about 2 Units, about 3 Units, about 4 Units, or about 5 Units per mg of therapeutic agent to about 10 Units, about 11 Units, about 12 Units, about 13 Units, about 14 Units, about 15 Units, about 16 Units, about 17 Units, about 18 Units, about 19 Units, or about 20 Units per mg of therapeutic agent. In some embodiments, the hyaluronidase is present in the compositions in an amount from about 1,000 Units, about 2,000 Units, about 3,000 Units, or about 4,000 Units per mg of the composition to about 5,000 Units, about 6,000 Units, about 7,000 Units, about 8,000 Units, about 9,000 Units, about 10,000 Units, or about 15,000 Units per mg of the composition. In some embodiments, hyaluronidase is present in the compositions of the invention in an amount from about 1,000 units, about 1,500 units, or about 2,000 units per mg of the composition to about 2,500 units, about 3,000 units, about 3,500 units, or about 4,000 units per mg of the composition.

[0109] In some embodiments, administering a therapeutic agent and particles comprising hyaluronidase to a subject results in at least about 90%, at least 95%, at least 99%, or at least 100% of the therapeutic agent, hyaluronidase, and / or particles dissolving within about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, or 40 minutes, to about 50 minutes, to about 60 minutes, to about 70 minutes, to about 80 minutes, to about 90 minutes, to about 100 minutes, to about 110 minutes, or to about 1 minute. In some embodiments, the therapeutic agent and particles comprising hyaluronidase are dissolved within 20 minutes, or within about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours, or more. In some embodiments, administering a therapeutic agent and particles comprising hyaluronidase to a subject results in at least about 90%, at least about 95%, at least 99%, or at least 100% of the therapeutic agent, hyaluronidase, and / or particles being dissolved within about 5, 10, 20, 30, or 40 minutes, to about 50 minutes, or to about 60 minutes, after administration to the subject. In some embodiments, administering particles comprising a therapeutic agent and hyaluronidase to a subject provides a therapeutic agent, hyaluronidase, and / or particles that are at least about 90%, at least about 95%, at least about 99%, or at least about 100% of the therapeutic agent, hyaluronidase, and / or particles within about 5, 10, 20, 30, or 40 minutes, to about 50, 60, 70, 80, 90, 100, 110, or 120 minutes after administration to the subject. The composition was dissolved within minutes, or within about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours, or more.

[0110] In some embodiments, by administering to a subject particles comprising a therapeutic agent and hyaluronidase, the method increases the distribution of the administered particles, therapeutic agent, and / or composition in the subject. For example, in some embodiments, the method increases the distribution of the particles, therapeutic agent, and / or composition under the skin of the subject. This can be determined by methods known to those skilled in the art, such as measuring the length of a lump, bleb, and / or wheal under the skin after administration. In some embodiments, a therapeutic agent administered to a subject according to the methods of the present invention can have improved 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 present invention.

[0111] In some embodiments, the methods of the present invention comprise administering to a subject a therapeutically effective amount of a therapeutic agent, hyaluronidase, particle, and / or composition of the present invention. As used herein, the term "therapeutically effective amount" refers to an amount of a therapeutic agent, hyaluronidase, particle, and / or composition of the present invention that elicits a therapeutically useful response in a subject. Those skilled in the art will understand that the therapeutic effect need not be complete or curative, as long as some benefit is provided to the subject.

[0112] As used herein, "treat," "treating," or "treatment" (and grammatical variations thereof) refers to any type of treatment that confers benefit to a subject and can mean that the severity of a subject's condition (e.g., a disease or disorder) is reduced, at least partially improved, or ameliorated, and / or some alleviation, reduction, or decrease in at least one clinical symptom associated with the subject's condition is achieved, and / or a delay in the progression of symptoms occurs. In some embodiments, the subject's condition can be reduced in the subject compared to the severity of the symptoms in the absence of the methods of the invention. In some embodiments, pain, swelling, and / or irritation at an injection site in a subject is reduced in the methods of the invention.

[0113] In some embodiments, the therapeutic agents, hyaluronidase, particles and / or compositions of the present invention may be administered in a therapeutically effective amount. As used herein, a "therapeutically effective amount" refers to an amount sufficient to treat a subject (as defined herein). Those skilled in the art will understand 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 may be achieved by administering a therapeutic agent, hyaluronidase, particle, or composition of the present invention.

[0114] As used herein, the terms "prevent," "preventing," and "prevention" (and grammatical variations thereof) refer to avoiding, alleviating, and / or delaying the onset of symptoms associated with a condition (e.g., a disease and / or disorder) and / or reducing the severity of the onset of symptoms associated with a condition compared to that which would occur in the absence of the methods of the invention. Prevention can be complete, e.g., the complete absence of symptoms. The prevention can also be partial, such that the onset and / or severity of the onset of symptoms in a subject is less than that which would occur in the absence of the methods of the invention. In some embodiments, the methods of the invention prevent or avoid pain, swelling, and / or irritation at an injection site in a subject.

[0115] In some embodiments, the therapeutic agents, hyaluronidase, particles, and / or compositions of the invention can be administered in a prevention effective amount. As used herein, a "therapeutically effective" amount is an amount sufficient to prevent symptoms associated with a condition (as defined herein) in a subject. Those skilled in the art will understand that the therapeutic effect need not be complete or curative, as long as some benefit is provided to the subject. In some embodiments, a prevention effective amount can be achieved by administering the therapeutic agents, hyaluronidase, particles, and / or compositions of the invention.

[0116] The present invention finds use in both veterinary and medical applications. Subjects suitable for treatment with the methods of the present invention include, but are not limited to, mammalian subjects. Mammals of the present invention 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 mammals in utero. Any mammal in need of treatment according to the present invention is suitable. Human subjects of both genders and at any stage of development (i.e., neonates, infants, juveniles, adolescents, and adults) can be treated according to the present invention. In some embodiments of the present invention, the subject is a mammal, and in some embodiments, the subject is a human.Human subjects include both males and females of all ages, including fetal subjects, newborn subjects, infant subjects, young subjects, adolescent subjects, adult subjects, and elderly subjects, as well as pregnant subjects.In certain embodiments of the present invention, the subject is a human adolescent and / or adult.

[0117] The methods of the invention may be carried out 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.

[0118] In some embodiments, the subject is "in need of" or "in need of" a method of the invention, e.g., the subject has a finding typically associated with a condition, is suspected of having a condition, and / or the subject has a condition.

[0119] In some embodiments, the methods of the present invention may provide increased dispersion, increased dissolution, and / or increased clearance of a therapeutic agent, including a therapeutic agent, vehicle, and / or infused particles, in the presence of hyaluronidase at a reduced (lower) concentration than the concentration of a hyaluronidase not in accordance with the present invention (e.g., a hyaluronidase not administered in the form of a particle of the present invention). For example, a method of the invention comprising administering particles of the invention comprising a first concentration of hyaluronidase may allow a composition comprising a therapeutic agent and / or the therapeutic agent to be administered to a subject and to be cleared and / or dissolved about one hour after administration of the therapeutic agent, whereas a method not in accordance with the invention (e.g., a method administering hyaluronidase that is not in a particle of the invention, and / or a method administering hyaluronidase dissolved in an aqueous solution, and / or a method that does not include and / or does not administer hyaluronidase) must use a higher hyaluronidase concentration than the first concentration to allow the same composition comprising the therapeutic agent and / or the therapeutic agent to be cleared and / or dissolved at the same time. In some embodiments, a method of the invention may use (e.g., administer to a subject) a reduced hyaluronidase concentration (e.g., at least about 5% or more) compared to a method not in accordance with the invention, and optionally obtain similar results (e.g., measurements within ±20%) to the methods of the invention. In some embodiments, the methods of the invention may use (e.g., administer to a subject) a reduced concentration of hyaluronidase compared to methods of administering free hyaluronidase and / or methods of administering hyaluronidase present in an aqueous composition, and optionally achieve results similar to those of the methods of the invention (e.g., measurements within ±20%).

[0120] In some embodiments, the methods of the invention may provide an increased (e.g., at least about 5%; e.g., at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or more) volume of therapeutic agent and / or composition comprising a therapeutic agent to be administered to a subject compared to methods not in accordance with the invention (e.g., methods that administer hyaluronidase that is not in particles of the invention, and / or methods that administer hyaluronidase dissolved in an aqueous solution, and / or methods that do not contain hyaluronidase and / or do not administer hyaluronidase), and optionally may achieve similar results (e.g., measurements within ±20%) or improved results compared to methods of the invention. Similar results may include similar dissolution profiles, dispersibility, bioavailability, swelling volume, side effects, pain, irritation at the injection site, etc. In some embodiments, the methods of the invention may provide an increased (e.g., at least about 5%; e.g., at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or more) volume of therapeutic agent or composition comprising the therapeutic agent compared to methods of administering free hyaluronidase and / or methods of administering hyaluronidase present in an aqueous composition, optionally achieving similar (e.g., measured within ±20%) or improved results compared to the methods of the invention.In some embodiments, the methods of the invention may provide a subject with an increased total administered volume (e.g., at least about 5%; e.g., at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or more) compared to methods of administering free hyaluronidase and / or methods of administering hyaluronidase present in an aqueous composition, optionally achieving similar results (e.g., measurements within ±20%) or improved results as the methods of the invention. In some embodiments, the methods of the invention may provide an increased (e.g., at least about 5%; e.g., at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or more) volume of a therapeutic agent or a composition comprising the therapeutic agent compared to a method in which hyaluronidase is not administered. In some embodiments, the methods of the invention may provide a subject with an increased total administered volume (e.g., at least about 5%; e.g., at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or more) compared to a method in which hyaluronidase is not administered.

[0121] In some embodiments, the methods of the invention may provide improved rates of dispersion, dissolution and / or clearance (e.g., at least about 5%; e.g., at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or more) of the therapeutic agent, composition comprising the therapeutic agent, vehicle and / or infused particle compared to methods not in accordance with the invention (e.g., methods that administer hyaluronidase that is not in a particle of the invention, and / or methods that administer hyaluronidase dissolved in an aqueous solution, and / or methods that do not contain and / or do not administer hyaluronidase). In some embodiments, an enhanced rate of dispersion, dissolution, and / or clearance (e.g., at least about 5%; e.g., at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or more) of the therapeutic agent, composition comprising the therapeutic agent, vehicle, and / or infused particle may be provided compared to a composition that does not contain the particles of the invention (e.g., a method of administering hyaluronidase that is not in a particle of the invention and / or a method of administering hyaluronidase dissolved in an aqueous solution).

[0122] In some embodiments, the methods of the invention may administer to a subject an increased (e.g., at least about 5%; e.g., at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or more) total administered volume of a composition (e.g., a non-aqueous formulation) compared to methods of administering free hyaluronidase and / or methods of administering hyaluronidase dissolved in an aqueous solution, optionally achieving similar (e.g., measured within ±20%) or improved results compared to the methods of the invention. In some embodiments, a composition comprising particles of the invention may allow an increased (e.g., at least about 5%; e.g., at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or more) total administered volume of the composition (e.g., non-aqueous formulation) to be administered to a subject compared to a composition that does not contain particles of the invention.

[0123] In some embodiments, compositions of the invention comprising hyaluronidase particles of the invention may provide an increased injection volume of the composition compared to the injection volume of a non-aqueous composition not comprising particles of the invention. In some embodiments, compositions of the invention (e.g., compositions comprising particles of the invention comprising hyaluronidase) may provide reduced injection pressure and / or in-line pressure during injection of the composition compared to the injection pressure and / or in-line pressure of a non-aqueous composition not comprising particles of the invention. In some embodiments, compositions of the invention comprising hyaluronidase particles of the invention may provide increased dispersion and / or absorption of one or more components of the composition (e.g., increased dispersion and / or absorption of solvent, therapeutic agent, and / or particles) compared to the dispersion and / or absorption of one or more components in a non-aqueous composition not comprising particles of the invention. In some embodiments, compositions of the invention comprising hyaluronidase particles of the invention may provide increased stability of one or more components in the composition compared to the stability of one or more components in a non-aqueous composition not comprising particles of the invention.

[0124] The invention will now be illustrated in the following non-limiting examples.

[0125] Example

[0126] Example 1

[0127] Using a small-scale microvitrification method, particles containing hyaluronidase type 5 (SIGMA H6254-500MG lot SLCK4168) and sucrose (SIGMA 1.00892.1003 lot K52895792 03) were prepared in 17 mM Trizma buffer (SIGMA T1503-500G lot 068K5451) with or without bovine gamma globulin (RMBIO BGG-BBZ-01K lot 20150424IG). Three stock solutions were used: The first stock contained no hyaluronidase but 21 mg / g sucrose and 77 mg / g bovine gamma globulin. The second stock contained 21 mg / g sucrose, 71 mg / g bovine gamma globulin, and 10 mg / g hyaluronidase. The third stock contained 20 mg / g sucrose, no bovine gamma globulin, and 80 mg / g hyaluronidase. Particles were prepared by mixing 10–12 microliters of hyaluronidase stock with 1 milliliter of dry n-octanol (f value = 0.3). Particles were formed by vortexing with a homogenizer (IKA T-10) at setting 6 for 30 seconds. 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 by adding n-pentanol, mixing, centrifuging (same settings as above), and then carefully removing the supernatant. The particles were dried under vacuum overnight and then sealed and stored at 5°C. The particles were spherical in shape and tested to measure enzyme activity.

[0128] Example 2

[0129] Dry, dense particles containing hyaluronidase, sucrose, and bovine gamma globulin (BGG) were produced using a microvitrification process. Three batches were produced. The resulting particles contained approximately 2% hyaluronidase by weight.

[0130] For the first batch, a protein stock solution was prepared at a total solids concentration of approximately 100 mg / ml. Octanol and aqueous stock were fed into an inline homogenizer (IKA T-25) equipped with a custom feed at a total flow rate of 176 ml / min (2.228 ml / min of which was aqueous). Approximately 10 ml of aqueous stock solution was processed. The resulting suspension was simultaneously fed into a 0.4 micron filter to recover the particles. The particles were then washed on the filter with pentanol and dried under nitrogen. The powder contained 1.1 wt% residual moisture, 0.1 wt% residual pentanol, and 0.5 wt% residual octanol. 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, and the largest particle measured was 8.5 microns.

[0131] For the second batch, a protein stock solution was prepared at a total solids concentration of approximately 102 mg / ml. Octanol and aqueous stock were fed into an in-line homogenizer (IKA T-25) equipped with a custom feed at rates of 156 ml / min and 1.963 ml / min, respectively. Approximately 99 ml of the aqueous stock solution was processed. The resulting suspension was then fed into a 0.4 micron filter to recover the particles. Filtration was performed at 10 atmospheres of pressure applied by compressed nitrogen. The particles were then washed on the filter with pentanol and dried under nitrogen. The powder contained 0.9 wt% residual moisture, 0.1 wt% residual pentanol, and 0.7 wt% residual octanol. 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, and the largest particle measured was 9.9 microns.

[0132] For the third batch, a protein stock solution was prepared with a total solids concentration of approximately 103 mg / ml. Octanol and aqueous stock were fed into an in-line homogenizer (IKA T-25) equipped with a custom feed at rates of 155 ml / min and 1.946 ml / min, respectively. Approximately 110 ml of aqueous stock solution was processed. The resulting suspension was then fed into a 0.4 micron filter to recover the particles. Filtering was performed at 10 atmospheres of pressure applied by compressed nitrogen. The particles were then washed on the filter with pentanol and dried under nitrogen. The powder contained 1.5 wt% residual moisture, 0.1 wt% residual pentanol, and 0.7 wt% residual octanol. 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, and the largest particle measured was 10.1 microns.

[0133] Example 3

[0134] In vitro release studies were performed and compared by using suspensions of microvitrified protein particles in either phosphate-buffered saline (PBS) containing 0.25 mg / mL hyaluronidase (PBS-Hyal medium) or PBS containing 0.5% hyaluronic acid and 0.25 mg / mL hyaluronidase (PBS-HA-Hyal medium). A release medium containing PBS and 0.25 mg / mL hyaluronidase was also included as a control. Particle suspensions contained bovine gamma globulin (BGG) and sucrose and were prepared in benzyl benzoate at a total solids concentration of 500 mg / mL. Approximately 10 μL of this suspension was added to 5 mL tubes containing either PBS-Hyal or PBS-HA-Hyal release medium.

[0135] The release medium containing the particle suspension was sampled over time, and the absorbance at 280 nm of the sampled release medium was measured to determine the amount of BGG released from the suspension over time after contact with the release medium (e.g., the concentration of BGG released from the particles and / or suspension and / or the concentration of BGG dissolved in the release medium after contact with the release medium). As can be seen from Figure 3, the sample containing the PBS-HA-containing release medium showed a slow release of BGG from the suspension over time, while the sample containing the PBS-HA-Hyal release medium, as seen in Figure 1, showed a rapid release of BGG from the suspension over time. The results of this experiment demonstrated the feasibility of using hyaluronidase in the aqueous phase to increase the dissolution rate of solidified proteins from non-aqueous suspensions, such as in the subcutaneous space.

[0136] Example 4

[0137] For three individual samples, suspensions of protein particles were tested for in vitro release in solutions of PBS (PBS medium-1, PBS medium-2, or PBS medium-3) or 0.5% hyaluronic acid (HA) in PBS (PBS-HA medium-1, PBS-HA medium-2, or PBS-HA medium-3) and compared. The in vitro release of suspensions of protein particles in PBS or HA was also compared to the in vitro release of suspensions of particles containing the same protein and hyaluronidase in PBS or HA.

[0138] A suspension of microvitrified particles containing bovine gamma globulin (BGG) and sucrose was prepared in benzyl benzoate at a total solids concentration of 500 mg / mL. Approximately 10 μL of this suspension was added to a 5 mL tube with PBS or HA as the release medium. The release medium containing the BGG particle suspension was sampled over time, and the absorbance of the sampled release medium at 280 nm was measured to determine the protein concentration in the release medium over time. Triplicate samples were prepared for each release medium used.

[0139] Figure 2 shows the measured absorbance over time for each of the three PBS medium samples, and Figure 3 shows the measured absorbance over time for each of the three PBS-HA medium samples. In Figures 2 and 3, the maximum measured absorbance indicates the time point for complete release of protein (BGG) from the suspension. As shown in Figure 2, the suspension added to PBS medium showed rapid release of protein over time, with complete release of protein completed within one hour. The suspension added to 0.5% hyaluronic acid (HA) release medium showed slower release, with complete release of protein occurring over several days, as shown in Figure 3.

[0140] A suspension of microvitrified particles containing hyaluronidase and BGG was prepared from a solution containing hyaluronidase at 2% by weight of the dissolved solids and BGG at approximately 98% by weight of the dissolved solids. The particles were prepared in the suspension in benzyl benzoate at a concentration of approximately 500 mg / mL of total solids. The same experiment as above was repeated, except that approximately 10 μL of the suspension containing particles containing BGG and hyaluronidase was added to a 5 mL tube with PBS or HA as the release medium. Assuming that hyaluronidase was completely released from the particles, there would be 0.025 mg of hyaluronidase per mL of the release medium, which is 10-fold less than the amount of hyaluronidase in Example 3. The release medium was sampled over time in the same manner as above, and the absorbance of the sampled release medium at 280 nm was measured to determine the protein concentration in the release medium over time. Three samples were prepared for each release medium used.

[0141] Figure 4 shows the measured absorbance over time for each of the PBS samples (PBS medium-1, PBS medium-2, or PBS medium-3) containing particles comprising BGG and hyaluronidase, and Figure 5 shows the measured absorbance over time for each of the three PBS-HA medium samples (PBS-HA medium-1, PBS-HA medium-2, or PBS-HA medium-3) containing particles comprising BGG and hyaluronidase. In Figures 4 and 5, the maximum measured absorbance indicates the time for complete release of the protein from the suspension. As shown in Figures 4 and 5, protein release from the suspension was complete within one hour for both the PBS and PBS-HA release media. These results indicate that the hyaluronidase contained within the solid protein particles efficiently degraded hyaluronic acid in the HA release media, allowing the protein to dissolve more quickly from the suspension into the aqueous layer of the release media. In addition, the amount of hyaluronidase in the particles prepared in Example 4 was approximately 25-fold lower than the amount of hyaluronidase added to the vehicle in Example 3, indicating that less hyaluronidase in particle form is required to increase protein release from the suspension compared to free hyaluronidase in solution.

[0142] Example 5

[0143] Protein release from suspensions containing solid particles with different amounts of hyaluronidase is compared. Particles having different amounts of hyaluronidase in the particles (e.g., 0.1%, 0.5%, 1%, 3%, or 4% by weight of the particles) can be prepared and can be prepared as described in one or more of Examples 1-4.

[0144] Example 6

[0145] Protein release from suspensions containing hyaluronidase particles of the present invention is compared to that from individual, solid protein particles. Particles containing different amounts of hyaluronidase (e.g., hyaluronidase in amounts of 0.1%, 0.5%, 1%, 3%, or 4% by weight of the particles) are prepared and may be prepared as described in one or more of Examples 1-4. Hyaluronidase particles are provided in suspensions with particles that do not contain hyaluronidase, and different concentrations of hyaluronidase are prepared. For example, suspensions containing hyaluronidase particles present at 0.1%, 0.5%, 1%, 3%, or 4% by weight of the total solids content of the suspension and suspensions containing particles that do not contain hyaluronidase may be prepared.

[0146] Example 7

[0147] The enzymatic activity of the hyaluronidase-containing solid protein particles was measured over time (e.g., by comparing turbidity to a standard curve for hyaluronidase EC 3.2.1.35 (HAse) using the method in Dorfman, A. (1955) Methods in Enzymology, Volume I, pp. 166-173) and compared to the activity of hyaluronidase in a stock formulation. Hyaluronidase-containing particles were prepared in formulations as shown in Table 1.

[0148] [Table 1]

[0149] Powders of the particle formulation were subjected to a 3-month stability test at 2-8°C or 25°C. At predetermined time points (initial (t=0), 30 days, and 90 days), the powders were rehydrated (20 mM sodium phosphate, pH 7.0, 37°C, 77 mM sodium chloride, and 0.01% (w / v) bovine serum albumin), and enzyme activity was measured as described in Dorfman, A. (1955) Methods in Enzymology, Volume I, pp. 166-173. The results are shown in Figures 6 and 7.

[0150] Example 8

[0151] Using the Gottingen minipig model, injection site reactions and pharmacokinetics are compared for an injectable suspension in a non-aqueous vehicle with or without hyaluronidase particles of the present invention and an injectable suspension containing microvitrified particles containing a therapeutic agent, with or without hyaluronidase.

[0152] Formulations were prepared according to the table below (BB = benzyl benzoate; EO = ethyl oleate).

[0153] [Table 1A]

[0154] The animals used were male Göttingen minipigs aged 7 to 8 months. 2 ml of each formulation was injected intraperitoneally over approximately 10 seconds using a 27G needle (4 groups per animal, 4 animals per group, a total of 8 animals). Local changes at the injection site (warmth, redness, swelling, and bleb size) were scored immediately after injection, 1 hour, 2 hours, 3 hours, 4 hours, and 6 hours after injection, and once daily for 14 days after injection.

[0155] Example 9

[0156] Dry, dense particles containing hyaluronidase, sucrose, and bovine serum albumin (BSA) were produced using a microvitrification process and compared with BSA particles without hyaluronidase. Two batches were produced. The resulting enzyme-containing particles contained approximately 0.37% hyaluronidase by weight of the particle.

[0157] For the first batch, a stock solution of BSA and sucrose was prepared at a total solids concentration of approximately 85 mg / ml. Octanol and aqueous stock were fed into an in-line homogenizer (IKA T-25) to produce discrete, spherical solid particles. Approximately 33 ml of the aqueous stock solution was processed. The resulting suspension was washed with solvent, filtered, and dried under nitrogen. The powder contained 3.2 wt.% residual moisture, 0.3 wt.% residual wash solvent, and 5.2 wt.% residual octanol. 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, and the largest particle measured was 11.9 microns.

[0158] For the second batch, a stock solution of BSA, sucrose, and hyaluronidase was prepared at a total solids concentration of approximately 85 mg / ml. Octanol and the aqueous stock were fed into an in-line homogenizer (IKA T-25) to produce discrete, spherical solid particles. Approximately 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% by weight residual moisture, 0.2% by weight residual washing solvent, and 5.3% by weight residual octanol. 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, and the largest particle measured was 10.7 microns.

[0159] The powder was subjected to stability testing for one month at 2-8°C, 25°C, and 40°C. At the designated time points, the powder was rehydrated and enzyme activity was measured as described in Dorfman, A. (1955) Methods in Enzymology, Volume I, 166-173 (Figure 8), and BSA aggregation was measured by SEC-HPLC (Figure 9).

[0160] Example 10

[0161] Dry, dense particles containing hyaluronidase, trehalose, and a monoclonal antibody (mAb) were fabricated using a microvitrification process and compared with mAb particles without hyaluronidase. Two batches were fabricated. The enzyme-containing particles contained approximately 0.3% hyaluronidase by weight of the particle.

[0162] For the first batch, a stock solution of mAb and trehalose was prepared at a total solids concentration of approximately 101 mg / ml. Octanol and aqueous stock were fed into an in-line homogenizer (IKA T-25) to produce 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 1.8% by weight residual moisture, 0.1% by weight residual washing solvent, and 0.5% by weight residual octanol. 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, and the largest particle measured was 8.9 microns.

[0163] For the second batch, a stock solution of mAb, hyaluronidase, and trehalose was prepared at a total solids concentration of approximately 100 mg / ml. Octanol and aqueous stock were fed into an in-line homogenizer (IKA T-25) to produce 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% by weight residual moisture, 0.1% by weight residual washing solvent, and 0.4% by weight residual octanol. 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, and the largest particle measured was 16.1 microns (Figure 10).

[0164] The powders were subjected to stability testing for 60 days at 2-8° C., 25° C., and 40° C. After 0, 15, and 30 days, the powders were rehydrated and the percent monomer of the mAb was measured via SEC-HPLC (FIGS. 11, 12, 13, 14, 15, and 16), and the enzyme activity was measured as described in Dorfman, A. (1955) Methods in Enzymology, Volume 1, 166-173 (FIG. 17).

[0165] Example 11

[0166] In contrast to Example 10, particles were produced using a microbatch microglassification process. In this process, approximately 12 μL of aqueous stock solution was processed for each sample. Particle formulations containing mAb, hyaluronidase, and various excipients (Table 2) were formed and stability tests were performed at 40°C. After 12 days, the powder was reconstituted, and enzyme activity was assayed as described in Dorfman, A. (1955) Methods in Enzymology, Volume I, pp. 166-173, and the aggregation state of the mAb was measured using SEC-HPLC (Table 3).

[0167] [Table 2]

[0168] [Table 3]

[0169] Example 12

[0170] Hyaluronidase nanoparticles (e.g., particles having an average size of about 100 nm to about 500 nm) are produced by adding an aqueous solution containing about 1 to 5 mg / ml of hyaluronidase to a dehydrating solvent using the methods described above. The powder is assayed for activity using one of the methods described above.

[0171] The foregoing is illustrative of the present invention and is not intended to limit the scope of the present invention. The present invention is defined by the appended claims, with equivalents of those claims being included within the scope of the present invention. All publications, patent applications, patents, patent publications, and other references cited herein are incorporated by reference in their entirety for the content relevant to the sentence and / or paragraph in which the reference is made.

Claims

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

9. The particles.

2. The particle of claim 1, wherein the therapeutic agent is a biological agent, optionally wherein the biological agent 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. 4. The particle of any one of claims 1 to 3, wherein the particle further comprises 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., albumin, e.g., serum albumin).

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

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

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

9. 9. The particle of any one of claims 1 to 8, wherein the hyaluronidase is present in the particle in an amount of from about 0.1%, about 0.5%, or about 1% by weight of the particle to about 2%, about 3%, about 4%, or about 5% by weight.

10. 10. The particle of any one of claims 1-9, wherein the therapeutic agent is present in the particle in an amount of from about 50%, about 55%, about 60%, about 65%, or about 70% by weight of the particle to about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% by weight of the particle, optionally wherein the therapeutic agent is present in the particle in an amount of from about 75% or about 80% by weight to about 85%, about 90%, or about 95% by weight of the particle.

11. The particle of any one of claims 1 to 10, wherein the hyaluronidase and the therapeutic agent are not covalently linked.

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

9. The particles.

13. 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 the group consisting of sucrose, glucose, trehalose, mannitol, sorbitol, dextrose, maltose, lactose, and any combination thereof.

14. 13. 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. 13. The particle of claim 12, wherein the stabilizer is albumin, optionally serum albumin.

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

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

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

19. The particle according to any one of claims 1 to 18, wherein the particle is spherical.

20. The particles have a density of at least about 0.5 g / cm 3 , at least about 0.6 g / cm 3 , at least about 0.7 g / cm 3 , at least about 0.8 g / cm 3 , at least about 0.9 g / cm 3 or at least about 1 g / cm 3 From this, it is approximately 1.1 g / cm 3 , about 1.2g / cm 3 , about 1.3g / cm 3 , approximately 1.4 g / cm 3 , about 1.5g / cm 3 , about 1.6g / cm 3 , about 1.7g / cm 3 , about 1.8g / cm 3 , about 1.9g / cm 3 or about 2 g / cm 3 20. The particle of any one of claims 1 to 19, having a density of up to 1000 .mu.m.

21. 21. The particle of any one of claims 1 to 20, 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. 22. The particles of any one of claims 1 to 21, wherein the particles have a total moisture content of less than about 3% of the total particle mass, optionally wherein the particles have a total moisture content of less than about 2% of the total particle mass.

23. 23. The particle of any one of claims 1 to 22, wherein the particle has a residual (e.g., octanol and / or pentanol) content in an amount of less than about 3% by weight of the total particle mass.

24. 24. The particle of any one of claims 1 to 23, wherein the particle is amorphous and / or the hyaluronidase in the particle is amorphous.

25. The particle according to any one of claims 1 to 24, wherein the particle is a microparticle or a nanoparticle.

26. 26. The particle of any one of claims 1 to 25, 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 free hyaluronidase, and 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 free hyaluronidase.

27. 27. The particle of any one of claims 1 to 26, wherein the enzymatic activity of the hyaluronidase after storage at about 4°C to about 40°C for at least about 3 months is within about ±50% of the enzymatic activity of free hyaluronidase, and optionally, wherein the enzymatic activity of the hyaluronidase after storage at about 4°C to about 40°C for at least about 3 months is within about ±40% of the enzymatic activity of free hyaluronidase.

28. The particle of any one of claims 1 to 27, wherein the hyaluronidase is hyaluronidase V.

29. A composition comprising particles according to any one of claims 1 to 28.

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

31. The composition is heated at a temperature of about 20 degrees Celsius to about 25 degrees Celsius and for about 1,000 seconds. -1 ~About 3,000s -1 31. The composition of claim 29 or 30, having a viscosity of about 20 centipoise (cP) to about 200 cP when measured at a shear rate of 100 psi.

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

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

34. The composition of any one of claims 29 to 33, wherein the composition further comprises a solvent, optionally wherein the solvent is an ester (e.g., an alkyl ester or an aryl ester).

35. The solvent may be benzyl benzoate, ethyl oleate, triglyceride esters (e.g., MIGLYOL 登録商標 812), ethyl lactate, sesame oil, and any combination thereof.

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

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

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

39. A composition comprising: a solvent; and Hyaluronidase-containing particles Including, wherein the particles have a moisture content of less than about 15% by weight of the particles and / or a water activity of less than about 0.

9. The composition.

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

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

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

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

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

45. The composition is heated at a temperature of about 20 degrees Celsius to about 25 degrees Celsius and for about 1,000 seconds. -1 ~About 3,000s -1 45. The composition of any one of claims 39 to 44, having a viscosity of about 20 centipoise (cP) to about 200 cP when measured at a shear rate of 100 psi.

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

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

48. 1. A method of increasing the dissolution of a therapeutic agent in a liquid, comprising: combining the liquid with the therapeutic agent and particles comprising hyaluronidase, the particles having 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 dissolution of the therapeutic agent in the liquid. The method comprising:

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

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

51. The method of claim 48, wherein the particles are particles according to any one of claims 1 to 28.

52. 49. 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 to 47.

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

54. 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 (e.g., added) to the liquid sequentially.

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

56. 56. The method of any one of claims 48-55, wherein dissolution of the therapeutic agent is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 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. 57. The method of any one of claims 48-56, wherein at least about 90%, at least about 95%, at least about 99%, or at least about 100% of the therapeutic agent is dissolved within about 20 minutes, about 30 minutes, or about 40 minutes to about 100 minutes, about 110 minutes, or about 120 minutes following combining the particles, the therapeutic agent, and the liquid, optionally wherein at least about 90%, at least about 95%, at least about 99%, or at least about 100% of the therapeutic agent is dissolved within about 20 minutes, about 30 minutes, or about 40 minutes to about 50 minutes, about 60 minutes, about 70 minutes, or about 80 minutes following combining the particles, the therapeutic agent, and the liquid.

58. 1. A method for improving tolerance and / or increasing absorption of a therapeutic agent by subcutaneous, intramuscular, and / or intradermal administration to a subject, comprising: administering to the subject subcutaneously, intramuscularly, and / or intradermally particles comprising hyaluronidase, 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 tolerance and / or increasing absorption of the therapeutic agent upon subcutaneous, intramuscular, and / or intradermal administration to the subject. The method comprising:

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

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

61. 59. The method of claim 58, wherein the particles are particles according to any one of claims 1 to 28.

62. 59. The method of claim 58, wherein said subcutaneously, intramuscularly and / or intradermally administering comprises administering to said subject a composition comprising said particles subcutaneously, intramuscularly and / or intradermally, optionally wherein said composition is a composition according to any one of claims 29 to 47.

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

64. 64. The method of any one of claims 58 to 63, wherein the method increases the dispersion of the particles or composition under the skin of the subject after the subcutaneous, intramuscular and / or intradermal administration.

65. 65. The method of any one of claims 58 to 64, wherein the method increases the absorption, bioavailability, distribution, metabolism and / or excretion of the therapeutic agent in the subject following subcutaneous, intramuscular and / or intradermal administration compared to the 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. 66. The method of any one of claims 58 to 65, wherein said administering subcutaneously, intramuscularly and / or intradermally comprises administering to said subject said particles or said composition subcutaneously, intramuscularly and / or intradermally in a volume of about 0.1 mL or about 0.5 mL to about 1 mL, about 2 mL, about 5 mL or about 10 mL.

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

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

69. 69. The method of any one of claims 58-68, wherein the administering subcutaneously, intramuscularly, and / or intradermally comprises separately administering the hyaluronidase and the therapeutic agent subcutaneously, intramuscularly, and / or intradermally.

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

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

72. 69. The method of any one of claims 58-68, wherein the administering subcutaneously, intramuscularly, and / or intradermally comprises administering the hyaluronidase and the therapeutic agent simultaneously to the subject, optionally wherein the hyaluronidase and the therapeutic agent are present in the same particle or the same composition.

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

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

75. 75. The method of any one of claims 58-74, wherein said administering subcutaneously, intramuscularly and / or intradermally comprises administering said particles and said 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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