Particles and suspensions containing hyaluronane degrading agents, and methods for using them.

Abstract

This disclosure provides stable, injectable particle suspensions of therapeutic biological agents and hyaluronane degraders that, in various embodiments, enable administration without the need for resuspension. In particular, the compositions disclosed herein are high-concentration, low-volume injectable particle suspensions that increase the bioavailability of therapeutic biological agents.

Description

[Technical Field]

[0001] Related applications This application claims the benefit of U.S. Provisional Application No. 63 / 581,549, filed on 8 September 2023. The entire teachings of the above application(s) are incorporated herein by reference.

[0002] Embedding by referencing data in XML This application incorporates, by reference, the sequence listing contained in the following eXtensible Markup Language (XML) file filed concurrently with this application. a) File name: 57261009-002_SL.xml, creation date: September 6, 2024, size: 2,365 bytes.

[0003] This disclosure relates to compositions and methods useful for the subcutaneous delivery of biopharmaceutical products for therapeutic purposes. In particular, the compositions and methods disclosed herein are stable, high-concentration, low-volume, and low-syringe-extraction-force injectable particle suspensions of therapeutic biological agents and hyaluronane degraders that can be administered to patients to increase the bioavailability of therapeutic biological agents. [Background technology]

[0004] Biological agents, particularly antibodies, have driven a paradigm shift in the drug discovery and development process over the past few decades, helping patients for whom there were previously few or no treatment options. For example, current monoclonal antibody (mAb) therapies often require large doses administered by intravenous (IV) infusion at high volume and low concentration, which can take several hours to deliver, causing patient discomfort and increasing the risk of infection. Subcutaneous (SC) injection offers a more desirable alternative for delivery, as it reduces the burden on hospitals and clinical facilities, requires less time, and lowers the risk of complications. However, SC injection of mAb particles often requires low delivery volumes, necessitating high concentrations that are difficult to achieve. The use of hyaluronane degraders allows for higher bioavailability of the active pharmaceutical ingredient. However, formulating hyaluronane degraders into particles presents challenges to their chemical and physical stability during processing and storage. Therefore, there is a need for stable particle compositions containing therapeutic biological agents and hyaluronane degraders. [Overview of the project]

[0005] Provided herein are particles or compositions comprising at least one therapeutic biological agent or a salt thereof, and a hyaluronan degrading agent.

[0006] In one embodiment, the present disclosure provides particles comprising at least one therapeutic biological agent or a salt thereof and a hyaluronan degrader.

[0007] In another aspect, the present disclosure relates to a pharmaceutically effective composition, The present invention provides a pharmaceutically effective composition comprising a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein substantially all of the particles comprise at least one therapeutic biological agent or a salt thereof and a hyaluronan degrading agent.

[0008] This disclosure also relates to a method for treating a disease or condition in a subject requiring treatment of the disease or condition, wherein the subject is given a pharmaceutically effective amount of a composition, The present invention provides a method for administering a pharmaceutically effective amount of a composition comprising a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein substantially all of the particles comprise at least one therapeutic biological agent or a salt thereof and a hyaluronan degrader.

[0009] In another aspect, the disclosure also relates to a pharmaceutically effective composition, The present invention provides a method for administering a pharmaceutically effective composition comprising a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein substantially all of the particles comprise at least one therapeutic biological agent or a salt thereof and a hyaluronan degrading agent.

[0010] This disclosure also relates to a syringe or portable drug delivery injection device and a composition, The present invention provides a kit comprising a composition comprising a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein substantially all of the particles comprise at least one therapeutic biological agent or a salt thereof and a hyaluronan degrader.

[0011] In some embodiments, this composition is useful for administering a suspension of high concentrations of therapeutic biological agent particles containing a hyaluronan degrader by syringe injection or portable drug delivery injection device without requiring manual pre-mixing or resuspension before administration.

[0012] A patent or application document must include at least one color drawing. A copy of this published patent or patent application with the color drawing(s) will be provided by the Patent Office upon request and payment of the required fees. [Brief explanation of the drawing]

[0013] [Figure 1]Shows an image of particles comprising hIgG (human immunoglobulin G) protein and hyaluronidase used in the compositions disclosed herein at a magnification of 4000×. [Figure 2] Shows a focused ion beam scanning electron microscope (FIB-SEM) image of smooth, spherical microparticles (e.g., as-is, sliced) with no void spaces, comprising trastuzumab and hyaluronidase. [Figure 3] Shows the particle sizes (D10, D50, D90) and span for trastuzumab, rituximab, mixed antibodies (trastuzumab and rituximab), and BSA suspensions. Non-settling suspensions with PS80 aggregating agent are also shown. Particle sizes and span are listed in units of μm. [Figure 4] Shows a plot depicting hyaluronidase activity in the particles (i.e., the amount of hyaluronic acid degraded (ug)) compared to the starting aqueous feed solution. [Figure 5] Shows the percentage digestion for the aqueous formulation (feed) relative to the suspension containing trastuzumab particles. The percentage digestion indicates the activity of hyaluronidase to degrade hyaluronic acid. Compared to the aqueous formulation, the suspension formulation had higher hyaluronidase stability at a storage temperature of 40°C over 3 months and 6 months. [Figure 6] Shows the percentage digestion for the aqueous formulation (feed) relative to the suspension containing particles of two therapeutic monoclonal antibodies (trastuzumab and rituximab). [Figure 7] Shows the percentage digestion for the aqueous formulation (feed) relative to the suspension containing BSA particles. [Figure 8] Shows the percentage of high molecular weight species (HMW%), sub-visible particles per mg (SVP >2um), and the percentage of charge variants (basic %, acidic %) obtained from microparticle suspensions containing BSA (with and without hyaluronidase) particles in PGD. [Figure 9]Trastuzumab samples with and without hyaluronidase exhibit similar levels of coagulation (i.e., HMW%) and fragmentation (%LMW). The percentage of trastuzumab monomer is also shown. [Figure 10] This demonstrates that trastuzumab samples with and without hyaluronidase exhibit similar charge variant profiles (i.e., percentages of acidic, neutral, and basic trastuzumab species). [Figure 11] The profiles of sub-visible particles (SVP >25 μm and >10 μm) from trastuzumab samples with and without hyaluronidase are shown. [Figure 12] The percentages of monomers and neutral species obtained from trastuzumab samples containing and without hyaluronidase are shown before and after storage at 4°C and 40°C for 3 and 6 months, respectively. [Figure 13] The cake height of the trastuzumab particle suspension is measured before and after storage at 25°C for 3 months, compared to a non-settling suspension (i.e., a suspension with PS80 flocculant). [Figure 14] This shows the measurement of the cake height of particles containing trastuzumab and rituximab in the suspension, compared to the non-sedimented suspension, before and after storage at 25°C for 48 hours. [Figure 15] The cake height of the trastuzumab particle suspension (containing hyaluronidase) is measured and compared to the non-settling suspension (i.e., the suspension containing PEG400 flocculant) after storage at 25°C for 3 days. [Figure 16] Graphs showing pharmacokinetic profiles for mAb and hyaluronidase microparticle suspensions (SC injection), as well as hyaluronidase-free Ab protein microparticle suspensions (SC injection), in a cohort of rats (male Wistars). [Figure 17] The plasma concentrations of the therapeutic protein trastuzumab in samples containing hyaluronidase (blue trace) compared to samples without hyaluronidase (red trace) are shown for both aqueous formulations and suspensions. [Figure 18A] This shows the degree of inflammation in miniature pigs injected with a suspension of trastuzumab particles, with and without hyaluronidase. Tissues are scored from 0 to 5 (0=absent; 1=minimal; 2=mild; 3=moderate; 4=noticeable; 5=severe) based on a severity scoring matrix. [Figure 18B] This shows the degree of inflammation in miniature pigs injected with trastuzumab aqueous formulations, with and without hyaluronidase. [Figure 18C] This shows the degree of fibrosis in miniature pigs injected with suspensions of trastuzumab particles, with and without hyaluronidase. [Figure 18D] This shows the degree of fibrosis in miniature pigs injected with trastuzumab aqueous formulations, with and without hyaluronidase. [Figure 18E] The mean total score of the suspension (including the standard error (SEM) of the mean) is shown. To generate the mean total score, the scores for inflammation and fibrosis observations for each animal were added together, and then the total scores of all animals in each group were averaged. [Figure 18F] This shows the average total score (including SEM) for aqueous formulations. [Figure 19A] This metabolic panel shows 25 serum analytes (only 4 are shown) that were tested a total of 36 times over 3 days (days 4, 8, and 15 after injection in miniature pigs) for both aqueous trastuzumab formulations and suspensions with and without hyaluronidase. [Figure 19B] The complete blood count (CBC) panels for both aqueous trastuzumab formulations and suspensions with and without hyaluronidase are shown, performed over 3 days (days 4, 8, and 15 after injection in miniature pigs). [Figure 20]Adding hyaluronidase to microparticles increased the bioavailability of the suspension in Sprague Dawley rats. Samples containing hyaluronidase (red trace) showed increased plasma concentrations of the therapeutic protein trastuzumab compared to formulations without hyaluronidase (blue trace). [Figure 21] The image shows a 500x magnification image of particles containing proteins and hyaluronidase used in the compositions disclosed herein. [Figure 22] The percentages of high molecular weight species (HMW%), sub-visible particles (>2 μm SVP) per 1 mg, and neutral species obtained from microparticle suspensions containing anticancer monoclonal antibodies (trastuzumab and rituximab) compared to aqueous formulations (supply) with and without hyaluronidase are shown at various time points (T0 before storage, after 3 months of storage at 4°C, and after 3 months of storage at 40°C). [Figure 23] The ratio of two anti-cancer monoclonal antibodies (trastuzumab and rituximab) co-formulated in microparticles is maintained by treatment, as measured by cation exchange chromatography (CEX). [Figure 24] This shows the stability of the protein (rituximab) in microparticles with various excipients (Arg.HCl, Arg.HCl and PS80, CaCl2, Arg.HCl and sucrose at pH 7.5, and sucrose and Arg.HCl) as measured by the levels of soluble coagulation (HMW%), charge variants (basic%), acidic%), and sub-visible particles (SVP >2 μm) per 1 mg. [Figure 25] This shows the stability of rituximab and hyaluronidase, as well as the hyaluronidase enzyme activity in microparticles containing various excipients, at various time points (before storage and 1 month after storage) (indicated by digestion percentage). The digestion percentage indicates the activity of hyaluronidase for digesting hyaluronic acid. [Figure 26]This shows the stability of rituximab in microparticles formulated with a pharmaceutically acceptable carrier liquid (ethyl oleate (EO), medium-chain triglycerides (MCT), MCT:EO in a 1:1 ratio, and propylene glycol diesters (PGD) of saturated vegetable fatty acids C8 and C10), as measured by the levels of soluble coagulation (HMW%), charge variants (basic%), and sub-visible particles (SVP >2 μm). [Figure 27] The enzyme activity is shown as digestibility in rituximab microparticles formulated in various pharmaceutically acceptable carrier liquids (ethyl oleate (EO), medium-chain triglycerides (MCT), MCT:EO in a 1:1 ratio, and propylene glycol diesters (PGD) of saturated vegetable fatty acids C8 and C10). [Figure 28] This shows the syringe extrusion force of trastuzumab particles formulated as a suspension in PGD (without PS80 flocculant) with and without hyaluronidase, and as a non-precipitating suspension in PGD or EO carrier liquid (with PS80 flocculant). [Figure 29] This shows the syringe extrusion force of suspensions of trastuzumab and mixed antibodies (trastuzumab + rituximab) with and without hyaluronidase (and suspensions with PS80 agglutinant). [Figure 30] This shows the syringe extrusion force of samples containing BSA particles formulated as a suspension in PGD, with and without hyaluronidase. [Figure 31] The image on the left shows the injection of a suspension consisting of hIgG particles in PGD using Schott (syriQ BioPure® (SCHOTT, Pharma)) syringe with a 1 / 2-inch 27g STW needle and BD (BD Neopak® (Becton, Dickinson and Company)) syringe with an 8mm 27g UTW needle, as well as a YpsoMate 2.25mL automated injector from Ypsomed. [Figure 32]The trastuzumab / hyaluronidase suspension containing 10 mg / ml of PEG400 as a flocculant (right image) did not settle after 3 days at room temperature, while the suspension without PEG400 (left image) settled and had a cake height of 73%. [Figure 33A] This shows a suspension of trastuzumab particles (533 mg / ml) in PGD containing hyaluronidase (20 U / mg protein) and propylene glycol (7.2 mg / ml) as a flocculant. [Figure 33B] This shows a suspension of trastuzumab particles (533 mg / ml) in PGD containing hyaluronidase (20 U / mg protein) and PEG300 (9.6 mg / ml) as an agglutinating agent. [Figure 33C] This shows a suspension of trastuzumab particles (533 mg / ml) in PGD, containing hyaluronidase (20 U / mg protein) and PEG600 (11.2 mg / ml) as a flocculant. [Figure 33D] This shows a suspension of trastuzumab particles (533 mg / ml) in PGD, containing hyaluronidase (20 U / mg protein) and ethylcellulose (5.4 mg / ml) as a flocculant. [Figure 34] This shows a suspension containing hIgG particles (400 mg / mL) with a flocculant of 5.5 mg / mL (PS80, PS80:PEG400 1:1, or PS80:propyene glycol 1:1). A control suspension was also prepared without the addition of a flocculant. [Figure 35] This shows a suspension containing hIgG particles (500 mg / mL) with a flocculant of 5.5 mg / mL (PS80, PS80:PEG400 1:1, or PS80:propien glycol 1:1). A control suspension was also prepared without the addition of a flocculant. [Figure 36]The injection profiles of a suspension containing hIgG particles (400 mg / mL) with a 5.5 mg / mL flocculant (PS80, PS80:PEG400 1:1, or PS80:propien glycol 1:1) and a control suspension without any flocculant are shown. [Figure 37] The injection profiles of a suspension containing hIgG particles (500 mg / mL) with a 5.5 mg / mL flocculant (PS80, PS80:PEG400 1:1, or PS80:propien glycol 1:1) and a control suspension without any flocculant are shown. [Modes for carrying out the invention]

[0014] Therapeutic biological agents, particularly monoclonal antibody (mAb) therapies, have dramatically improved the treatment of human diseases. However, the delivery of these biological agents is burdensome for patients. The standard of administration is often intravenous (IV) infusion at low concentrations, which can take several hours to deliver, causing patient discomfort and increasing the risk of infection. Subcutaneous (SC) delivery by simple injection is preferred, but the constraint on low SC delivery volumes (1.5–2.0 mL) necessitates antibody concentrations exceeding 100 mg / mL, which is often impractical. Solution concentrations above 100 mg / mL are highly viscous, leading to very high injection force, which often accelerates the degradation of the therapeutic antibody composition. The use of particle suspension technology with particles containing hyaluronane degraders allows for the delivery of greater volumes (more than 2.0 mL) of therapeutic biological products (e.g., antibodies) at concentrations greater than 500 mg / mL while preserving the complete structure and bioactivity of the therapeutic biological product (e.g., mAb). Therefore, by converting the delivery of therapeutic biological products from IV to SC, this technology can provide benefits to patients, healthcare providers, payers, and pharmaceutical developers. While the use of hyaluronane degraders enables the benefit of higher bioavailability of therapeutic biological products, formulating hyaluronane degraders into particles presents significant challenges to their chemical and physical stability during process and storage. Therefore, a stable particle composition containing both the therapeutic biological product and the hyaluronane degrader is required.

[0015] This disclosure relates, in general terms, to particles comprising at least one therapeutic biological agent or a salt thereof, and a hyaluronan degrading agent. In some embodiments, the particles contain less than about 25% internal void space. In certain embodiments, the roundness of the particles is about 0.80 to about 1.00.

[0016] This disclosure also relates to pharmaceutically effective compositions and methods comprising a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein substantially all of the particles comprise at least one therapeutic biological agent or a salt thereof and a hyaluronan degrader. In some embodiments, the concentration of the therapeutic biological agent or a salt thereof in the composition is greater than about 250 mg / mL. In some embodiments, the compositions and methods described herein further comprise a flocculant. In some embodiments, the compositions and methods described herein, the composition has a flocculation volume greater than about 60% after initial mixing. In certain embodiments, the composition has improved stability of the therapeutic biological agent and hyaluronan degrader compared to aqueous compositions comprising at least one therapeutic biological agent and a hyaluronan degrader in soluble form. In some embodiments, the composition is administered by syringe or portable drug delivery injection device. For a description of exemplary compositions of this disclosure comprising a plurality of particles comprising at least one therapeutic biological agent and a hyaluronan degrader suspended with a flocculant in a pharmaceutically acceptable liquid carrier, see Examples 1-2 herein.

[0017] It will be readily apparent that the aspects and embodiments generally described herein are illustrative. The following more detailed descriptions of various aspects and embodiments are not intended to limit the scope of this disclosure, but are merely representative of various aspects and embodiments. Furthermore, the compositions and methods disclosed herein can be modified by those skilled in the art without departing from the scope of this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. All publications and patents referenced herein are invoked by reference.

[0018] definition For the purposes of this disclosure, unless otherwise expressly stated, the following definitions will be used.

[0019] In the context describing this disclosure, the terms “a,” “an,” “the,” and similar reference subjects should be interpreted as encompassing both singular and plural forms unless otherwise indicated herein or unless the context clearly contradicts this. All compositions described herein may be performed in any preferred form unless otherwise indicated herein or unless the context clearly contradicts this. The use of any and all examples or exemplary language provided herein (e.g., “etc.”) is intended solely to better illustrate this disclosure and does not impose any limitation on the scope of the claimed disclosure. Nothing in this specification should be interpreted as indicating that any unclaimed element is essential to the implementation of this disclosure.

[0020] The term "approximately" in relation to a given numerical value, such as temperature or duration, means that the value includes a number that is within 10% of the specified value.

[0021] As used herein, the terms “administer” or “be administered” mean the actual physical introduction of the composition into or onto a subject, as appropriate. Any and all methods of introducing a composition into a subject are contemplated in accordance with this disclosure, and the composition is independent of, and should not be construed as dependent on, any particular means of introduction. Means of introduction are known to those skilled in the art and are illustrated herein.

[0022] As used herein, the “alkyl” group or “alkane” is a fully saturated linear or branched non-aromatic hydrocarbon. Typically, linear or branched alkyl groups have 1 to about 20 carbon atoms, preferably 1 to about 10 carbon atoms, unless otherwise defined. Examples of linear and branched alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, tert-pentyl, neo-pentyl, iso-pentyl, sec-pentyl, 3-pentyl, sec-iso-pentyl, active-pentyl, hexyl, heptyl, octyl, ethylhexyl, and the like. 1-8Linear or branched alkyl groups are also referred to as “lower alkyl” groups. Alkyl groups with two open valencies are sometimes referred to as alkylene groups, such as methylene, ethylene, and propylene. Furthermore, as used herein, in the examples and claims, the term “alkyl” (or “lower alkyl”) is intended to include both “unsubstituted alkyl” and “substituted alkyl,” the latter referring to an alkyl moiety having substituents that replace hydrogens on one or more carbons of a hydrocarbon backbone. Such substituents, unless otherwise specified, may include, for example, alkyl, halogen, hydroxyl, carbonyl (carboxyl, and alkoxycarbonyl, formyl, or acyl), thiocarbonyl (thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphine, amino, amide, amidine, imine, cyano, nitro, azide, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties. It will be understood by those skilled in the art that, where appropriate, the substituted moieties on the hydrocarbon chain themselves may be substituted. For example, substituents on substituted alkyls may include amino, azide, imino, amide, phosphoryl (including phosphonates and phosphinates), sulfonyl (including sulfate, sulfonamide, sulfamoyl, and sulfonate), and silyl groups, as well as ethers, alkylthio, carbonyl (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN, and other substituted and unsubstituted forms. Exemplary substituted alkyls are listed below. Cycloalkyls may be further substituted with alkyl, alkenyl, alkoxy, alkylthio, aminoalkyl, carbonyl-substituted alkyls, -CF3, -CN, and the like. In some embodiments, the term “alkyl” may mean “cycloalkyl” referring to a non-aromatic carbon ring having 3 to 10 carbon ring atoms, which are carbon atoms bonded together to form a ring. The ring may be saturated or may have one or more carbon-carbon double bonds.Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, and cycloheptyl, as well as crosslinked and caged saturated ring groups such as norbornyl and adamantyl. As described herein, organic solvents include, but are not limited to, aliphatic hydrocarbon solvents, aromatic hydrocarbon solvents, alcohols or alkyl alcohols, alkyl ethers, sulfoxides, alkyl ketones, alkyl acetates, trialkylamines, alkyl formates, trialkylamines, or combinations thereof. Aliphatic hydrocarbon solvents may include, but are not limited to, pentane, hexane, heptane, octane, cyclohexane, or combinations thereof. Aromatic hydrocarbon solvents may include, for example, benzene, toluene, or combinations thereof. Alcohols or alkyl alcohols include, for example, methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, decanol, amyl alcohols, or combinations thereof. Alkyl ethers include methyl, ethyl, propyl, and butyl ethers, such as diethyl ether, diisopropyl ether, or combinations thereof. Sulfoxides include dimethyl sulfoxide (DMSO), decyl methyl sulfoxide, tetradecyl methyl sulfoxide, or combinations thereof. The term "alkyl ketone" refers to ketones substituted with alkyl groups, such as acetone, ethyl methyl ketone, or combinations thereof. The term "alkyl acetate" refers to acetates substituted with alkyl groups, such as ethyl acetate, propyl acetate (n-propyl acetate, iso-propyl acetate), butyl acetate (n-butyl acetate, iso-butyl acetate, sec-butyl acetate, tert-butyl acetate), amyl acetate (n-pentyl acetate, tert-pentyl acetate, neo-pentyl acetate, iso-pentyl acetate, sec-pentyl acetate, 3-pentyl acetate, sec-iso-pentyl acetate, active-pentyl acetate), 2-ethylhexyl acetate, or combinations thereof.The term "alkylformate" refers to a formate substituted with an alkyl group, such as methylformate, ethylformate, propylformate, butylformate, or a combination thereof. The term "trialkylamine" refers to an amino group substituted with three alkyl groups, such as triethylamine.

[0023] As used herein, “amino acid” or “residue” means any naturally occurring or non-naturally occurring amino acid, any amino acid derivative, or any amino acid mimetic known in the art. This includes the L and D forms of each amino acid, but the L form is generally preferred. In some embodiments, the term refers to any one of the 20 naturally occurring amino acids: L forms of glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), cysteine ​​(Cys), methionine (Met), serine (Ser), threonine (Thr), glutamine (Gin), asparagine (Asn), glutamic acid (Glu), aspartic acid (Asp), lysine (Lys), histidine (His), arginine (Arg), phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr). In some embodiments, the amino acid side chain may be a side chain of Gly, Ala, Val, Leu, Ile, Met, Cys, Ser, Thr, Trp, Phe, Lys, Arg, His, Tyr, Asn, Gln, Asp, Glu, or Pro. In certain embodiments, the amino acid derivative is an amino acid salt, such as an amino acid salt of hydrochloric acid, phosphoric acid, DL-lactic acid / glycolic acid, succinic acid, citric acid, sulfuric acid, sodium hydroxide, potassium hydroxide, sodium succinate, sodium phosphate, sodium acetate, sodium citrate, or sodium sulfate.

[0024] Where used herein, unless the context requires otherwise, the term “comprise,” and its variations, such as “comprising,” “comprises,” and “comprised,” are not intended to exclude further additives, components, elements, or steps. The terms “including” and “comprising” may be used interchangeably. Where used herein, phrases such as “selected from the group consisting of,” and “selected from,” include mixtures of the specified materials. Where numerical limits or ranges are described herein, their endpoints are included. Furthermore, all values ​​and subranges within numerical limits or ranges are specifically included as if they were explicitly written herein. References to elements in the singular are intended to mean “one or more,” rather than “one and only one,” unless specifically stated otherwise. Unless specifically stated otherwise, terms such as “several” refer to one or more, and singular terms such as “a,” “an,” and “the” refer to one or more.

[0025] The term "oligopeptide" is used to refer to peptides with fewer amino acid members, as opposed to polypeptides or proteins. The oligopeptides described herein typically consist of about 2 to about 40 amino acid residues. Oligopeptides include dipeptides (2 amino acids), tripeptides (3 amino acids), tetrapeptides (4 amino acids), pentapeptides (5 amino acids), hexapeptides (6 amino acids), heptapeptides (7 amino acids), octapeptides (8 amino acids), nonapeptides (9 amino acids), decapeptides (10 amino acids), undecapeptides (11 amino acids), dodecapeptides (12 amino acids), eicosapeptides (20 amino acids), tricontapeptides (30 amino acids), tetracontapeptides (40 amino acids), and so on. Oligopeptides can also be classified according to their molecular structure: aeruginosine, cyanopeptrin, microcystin, micropyridine, microginine, anabaenopeptin, and cyclamide, etc. A homooligopeptide is an oligopeptide containing the same amino acids. In certain embodiments, the homooligopeptide includes polyvaline, polyalanine, and polyglycine hexamers of 10 amino acids.

[0026] The term "peptide" is defined as a small protein consisting of two or more amino acids, linked by the carboxyl group of one amino acid to the amino group of another amino acid. Therefore, at its basic level, any type of peptide synthesis involves repeated steps of adding amino acids or peptide molecules to each other or to an existing peptide chain. The term "peptide" generally refers to a peptide having approximately 2 to approximately 100 amino acids, while a polypeptide or protein has approximately 100 or more amino acids, up to the full-length sequence that can be translated from the gene. Additionally, as used herein, a peptide may be a partial sequence or portion of a polypeptide or protein. In a particular embodiment, the peptide is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, It consists of 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acid residues. In some embodiments, the peptide is about 30 to about 100 amino acid residues long.

[0027] As used herein, the term “pharmaceutically acceptable” means a composition that, within the bounds of sound medical judgment, is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, and that provides a reasonable benefit / risk ratio. Preferably, the term “pharmaceutically acceptable” means that it is approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally accepted pharmacopoeia for use in animals, more specifically in humans.

[0028] The term "protein" is defined as a linear polymer constructed from approximately 20 different amino acids. The type and sequence of amino acids in a protein are specified by the DNA that produces that protein. In certain embodiments, the sequence may be natural or unnatural. The sequence of amino acids determines the overall structure and function of the protein. In some embodiments, a protein may contain more than 50 residues. In certain embodiments, a protein may contain a length of more than approximately 101 residues. The net charge of a protein can be determined by two factors: 1) the total count of acidic amino acids versus basic amino acids, and 2) the specific solvent pH environment that exposes positive or negative residues. As used herein, a "net positively charged or net negatively charged protein" is a protein that has a net positive or net negative charge under non-denaturing pH conditions. Generally, those skilled in the art will recognize that all proteins, regardless of their amino acid composition, can be considered "net negatively charged proteins" depending on their pH and / or solvent environment. For example, different solvents can expose negative or positive side chains depending on the solvent pH. The protein is preferably selected from any type of enzyme or antibody or fragment thereof that exhibits substantially the same activity as the corresponding enzyme or antibody. The protein can function as a structural material (e.g., keratin), as an enzyme, as a hormone, as a transporter (e.g., hemoglobin), as an antibody, or as a regulator of gene expression. Proteins are required for the structure, function, and regulation of cells, tissues, and organs. In some embodiments, the protein is a therapeutic biological agent. In certain embodiments, the protein is bovine serum albumin (BSA) or human serum albumin (HSA).

[0029] As used herein, the term “substantially” means the majority or most of the matter, such as at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more.

[0030] It is understood that the specific order or hierarchy of steps in the disclosed process is illustrative of an exemplary approach. It is understood that the specific order or hierarchy of steps in the process may be rearranged based on design choices. Some steps may be performed simultaneously. The phrases “embodiments” do not imply that such embodiments apply to all configurations of the subject art. Disclosures relating to embodiments may apply to all embodiments or to one or more embodiments. The phrases “embodiments” may refer to one or more embodiments, or vice versa.

[0031] particle Unless otherwise defined, all technical terms, notations, and other scientific terms used herein are intended to have meanings generally understood by those skilled in the art to which this disclosure belongs. In some cases, terms having generally understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from those generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly used by those skilled in the art using conventional methodologies. Where appropriate, unless otherwise specified, procedures involving the use of commercially available kits and reagents are generally carried out according to the protocols and / or parameters defined by the manufacturer. Where used herein, the phrase "and / or" means that when used in a list of two or more items, any one of the listed items may be used alone or any combination of two or more of the listed items may be used. For example, if a composition is described as containing or excluding components A, B, and / or C, the composition may contain or exclude A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.

[0032] As used herein, the terms “particle” or “particles” or “microparticle” or “microparticles” are interchangeable in their broadest sense to refer to separate bodies or bodies. The particles described herein are circular and have a controlled dispersibility with characteristic sizes ranging from sub-micrometers to tens of micrometers, in contrast to, for example, the porous monolithic “cakes” typically produced during conventional freeze-drying. This form allows for a fluid powder (e.g., characterized by a low Hausner ratio) without post-processing. In some embodiments, the term “particle” refers to an amount of protein or proteins, e.g., a therapeutic biologic or therapeutic biologics, in either a state of matter that is substantially solid compared to a droplet. In some embodiments, the therapeutic biologic is hydrophobic. In some embodiments, the therapeutic biologic is hydrophobic and soluble in a vehicle (e.g., a non-aqueous solvent and / or aqueous solvent).

[0033] As disclosed herein, a therapeutic biological product, also known as a biopharmaceutical product or biopharmaceutical, is any pharmaceutical drug manufactured in, extracted from, or semi-synthesized from a biological source. Therapeutic biological products can include a wide range of products such as vaccines, blood and blood components, allergens, somatic cells, gene therapies, tissues, and recombinant therapeutic proteins. Biological products can be isolated from various natural sources, e.g., humans, animals, or microorganisms, and can be produced by biotechnological methods or other techniques. In some embodiments of this disclosure, the therapeutic biological product is an antibody or a fragment thereof. In some embodiments, the antibody is a mammalian or human antibody, e.g., bovine IgG, human IgG, or a monoclonal antibody (mAb). In certain embodiments, the antibody is rituximab or trastuzumab. In certain embodiments, the therapeutic biological product is a fragment of an antibody. In some embodiments, the compositions described herein comprise at least two plurality of particles (e.g., first and second plurality of particles) suspended in a pharmaceutically acceptable liquid carrier, each plurality of particles comprising at least one therapeutic biological agent (e.g., at least one different therapeutic biological agent compared to the particles in the other plurality). In some embodiments, the compositions described herein comprise at least two plurality of particles (e.g., first and second plurality of particles) suspended in a pharmaceutically acceptable liquid carrier, each plurality of particles comprising a hyaluronan degrader and at least one antibody, e.g., plurality of rituximab particles and plurality of trastuzumab particles.

[0034] The terms “antibody” and “immunoglobulin” are used interchangeably in their broadest sense and include monoclonal antibodies, polyclonal antibodies, multivalent antibodies, and multispecific antibodies, regardless of how they are produced, i.e., using immunization, recombination, or synthetic methodologies. Antibodies can be gamma globulin proteins found in the blood or other bodily fluids of vertebrates that function in the immune system to bind antigens and thus identify and / or neutralize foreign substances. Antibodies can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each having a heavy chain named alpha, delta, epsilon, gamma, and mu, respectively. The gamma class is further divided into subclasses based on differences in sequence and function, and for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In some embodiments of this disclosure, the IgG antibody is an IgG1 antibody. In certain embodiments of this disclosure, the IgG antibody is a monoclonal IgG antibody. The light chain from any vertebrate species can be assigned to one of two distinct types, e.g., kappa and lambda, based on the amino acid sequence of its constant domain.

[0035] The recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. In some embodiments, the light chain is classified as either kappa or lambda. In some embodiments, the heavy chain is classified as gamma, mu, alpha, delta, or epsilon, which then define the classes of immunoglobulins, IgG, IgM, IgA, IgD, and IgE, respectively. In certain embodiments of this disclosure, the antibody is an IgG antibody.

[0036] The exemplary antibody (immunoglobulin) structural unit is a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kD) and one "heavy" chain (approximately 50-70 kD). The N-terminus of each chain defines a variable region of approximately 100-110 or more amino acids, primarily involved in antigen recognition. The terms "variable light" chain, domain, region, and component are used interchangeably, "VL" or "V" L "VH" is an abbreviation for the light chain of an antibody or antibody fragment. Similarly, the terms "variable weight" chain, domain, region, and component are used interchangeably as "VH" or "VH". H Abbreviated as "Ig", it refers to the heavy chain of an antibody or antibody fragment. Antibodies are generally heterotetrameric glycoproteins composed of two identical light (L) chains and two identical heavy (H) chains. Each L chain is linked to an H chain by one covalent disulfide bond. The two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. The H and L chains define specific Ig domains. In particular, each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for the alpha and gamma chains, respectively, and four CH domains for the p and c isotypes. Each L chain has a variable domain (VL) at its N-terminus, followed by a constant domain (CL) at the other end. The VL aligns with the VH, and the CL aligns with the first constant domain (CHL) of the heavy chain. The constant domain contains the Fc region, which includes the carboxyl-terminal portions of both H chains held together by the disulfide. The effector function of antibodies such as ADCC is determined by the sequence within the Fc region, which is also the portion recognized by the Fc receptor (FcR) found on certain types of cells.

[0037] As disclosed herein, the pairing of VH and VL forms a “variable region” or “variable domain” containing the amino-terminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as “VH.” The variable domain of the light chain may be referred to as “VL.” The V domain contains an “antigen-binding site,” which influences antigen binding and defines the specificity of a particular antibody to its particular antigen. The V region consists of relatively invariant stretches called framework regions (FRs) (generally about 4) of 15-30 amino acids, separated by shorter, extremely variable regions called “hypervariable regions” (generally about 3), each generally 9-12 amino acid lengths, spanning about 110 amino acid residues. The FRs largely adopt the p-sheet configuration, and the hypervariable regions form loops that connect the p-sheet structure and, in some cases, form part of the p-sheet structure. In certain embodiments, “hypervariable regions” refer to regions of the antibody variable domain that are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies contain six hypervariable regions, three located in the VH region (H1, H2, H3) and three in the VL region (L1, L2, L3). "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as defined herein.

[0038] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used interchangeably herein to refer to an antibody in its substantially intact form, rather than as an antibody fragment as defined above. This term particularly refers to an antibody having a heavy chain containing an Fc region. A full-length antibody may be a natural sequence antibody or an antibody variant. In certain embodiments, an “intact” or “whole” antibody includes an antigen-binding site, as well as CL and at least heavy chain constant domains, CH1, CH2, and CH3. The constant domains may be natural sequence constant domains, e.g., human natural sequence constant domains, or amino acid sequence variants thereof.

[0039] As shown above, the term antibody as used herein, unless otherwise stated or if the context clearly contradicts it, includes fragments of antibodies that specifically interact with an antigen, for example, that retain the ability to bind to an antigen. It has been shown that the antigen-binding function of an antibody may be performed by a fragment of a full-length antibody. Examples of binding fragments encompassed within the term "antibody" include: (i) monovalent fragments consisting of Fab' or Fab fragments, light chain variable domain (VL), heavy chain variable domain (VH), light chain constant region (CL), and heavy chain constant region domain 1 (CH1) domain, or monovalent antibodies as described in WO2007 / 059782; (ii) bivalent fragments containing F(ab')2 fragments, two Fab fragments linked by disulfide crosslinks in the hinge region; (iii) Fd fragments essentially consisting of VH and CH1 domains; (iv) Fv fragments essentially consisting of VL and VH domains of a single arm of the antibody; (v) dAb fragments Ward et al., Nature 341, 544-546 (1989), which essentially consist of a VH domain and are also called domain antibodies Holt et al; Trends Biotechnol. 2003 November; 21(11): 484-90; and (vi) camelid antibodies or nanobodies Revets (vii) includes isolated complementarity-determining regions (CDRs). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombination methods by a synthetic linker that allows them to be constructed as a single protein chain where the VL and VH regions pair up to form a monovalent molecule (known as single-chain antibodies or single-chain Fv (scFv); see, for example, Revets et al; Expert Opin Biol Ther. 2005 January; 5(1): 111-24, and Bird et al., Science 242, 423-426 (1988)). Such single-chain antibodies are included within the scope of the term antibody unless otherwise specified or clearly indicated by the context.While such fragments are generally included within the meaning of antibodies, they are grouped together and each independently represents a unique feature of the present invention, exhibiting different biological properties and usefulness. These and other useful antibody fragments in the context of the present invention will be discussed further herein.

[0040] As disclosed herein, “whole antibody fragments containing a variable domain” include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. A “Fab fragment” consists of the entire L chain along with a variable region domain (VH) of the H chain and a first constant domain (CHI) of one heavy chain. Each Fab fragment is monovalent with respect to antigen binding, i.e., has a single antigen-binding site. A “Fab' fragment” differs from a Fab fragment by having a small number of additional residues at the carboxyl terminus of the CHI domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the herein nomenclature for Fab' having a free thiol group in the cysteine ​​residue(s) of the constant domain. An “F(ab')2 fragment” roughly corresponds to two disulfide-linked Fab fragments with bivalent antigen-binding activity and can still crosslink antigens. “Fv” is the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a dimer of one heavy chain variable domain and one light chain variable domain in a tight, non-covalent association. In the single-chain Fv (scFv) species, the one heavy chain variable domain and the one light chain variable domain can be covalently linked by a flexible peptide linker so that the light and heavy chains can associate in a “dimeric” structure similar to that of the double-chain Fv species. The folding of these two domains yields six hypervariable loops (three from the H and L chains) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. A “single-chain Fv,” also abbreviated as “sFv” or “scFv,” is an antibody fragment containing VH and VL antibody domains linked together to form a single polypeptide chain. In certain embodiments, the scFv polypeptide further includes a polypeptide linker between the VH and VL domains, which allows the scFv to form a desired structure for antigen binding. In some embodiments, the “single variable domain” is half of the Fv (containing only three antigen-specific CDRs) that has lower affinity than the entire binding site but still has the ability to recognize and bind to the antigen.

[0041] In some embodiments, a “diabody” refers to an antibody fragment having two antigen-binding sites, the fragment containing a heavy-chain variable domain (VH) attached to a light-chain variable domain (VL) on the same polypeptide chain (VH-VL). Smaller antibody fragments are prepared by constructing an sFv fragment with a short linker (about 5-10 residues) between the VH and VL domains so that inter-chain pairing rather than intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites. In some embodiments, a diabody may be bivalent or bispecific. In certain embodiments, a bispecific diabody is a heterodimer of two “crossover” sFv fragments, where the VH and VL domains of the two antibodies are on different polypeptide chains. Triabodies and tetrabodies are also generally known in the art.

[0042] The “antigen-binding fragments” of antibodies described herein consist of only a portion of an intact antibody and generally include the antigen-binding site of the intact antibody, and therefore retain the ability to bind to an antigen. Exemplary examples of antibody fragments encompassed by this definition include, but are not limited to, the following: (i) Fab fragments having VL, CL, VH, and CH1 domains; (ii) Fab' fragments which are Fab fragments having one or more cysteine ​​residues at the C-terminus of the CH1 domain; (iii) Fd fragments having VH and CH1 domains; (iv) Fd' fragments having VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (v) Fv fragments having VL and VH domains of a single arm of the antibody; (vi) fragments consisting of a VH domain. (vii) an isolated CDR region, (viii) a bivalent fragment comprising an F(ab')2 fragment and two Fab' fragments linked by disulfide crosslinking in the hinge region, (ix) a single-chain antibody molecule, e.g., single-chain Fv, scFv, (x) a "diabody" having two antigen-binding sites comprising a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain, and (xi) a "linear antibody" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) that, together with a complementary light-chain polypeptide, form a pair of antigen-binding sites. In some embodiments, the "antigen-binding site" generally refers to a molecule comprising at least a hypervariable region and a framework region required to confer antigen-binding function to the V domain. The antigen-binding site may be in the form of an antibody or antibody fragment (such as dAb, Fab, Fd, Fv, F(ab')2, or scFv) in the methods described herein. In some embodiments, the antigen-binding fragment competes for antigen binding with an intact antibody, such as an intact antibody from which the fragment originates.

[0043] The term “fragment” refers to a portion or part of an antibody or antibody chain that contains fewer amino acid residues than an intact or complete antibody or antibody chain. As used herein, the term “fragment” of an antibody molecule includes Fc fragments and antigen-binding fragments of an antibody, such as antibody light chain variable domains (VL), antibody heavy chain variable domains (VH), single-chain antibodies (scFv), F(ab')2 fragments, Fab fragments, Fd fragments, Fv fragments, single-domain antibody fragments (DAb), one-armed (monovalent) antibodies, or any antigen-binding molecule formed by combinations, assemblies, or conjugations of such antigen-binding fragments.

[0044] In some embodiments, the terms “single-chain Fv” or “scFv” or “single-chain” antibody may refer to an antibody fragment containing the VH and VL domains of the antibody, where these domains reside on a single polypeptide chain. Generally, the Fv polypeptide further includes a polypeptide linker between the VH and VL domains, which allows the sFv to form a desired structure for antigen binding. For a review of sFv, see Pluckthun, THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269–315 (1994).

[0045] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting that population are identical except for naturally occurring mutations that may be present in small amounts. Monoclonal antibodies (mAbs) are highly specific and directed toward a single antigenic site or determinant on an antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized without contamination by other antibodies. Monoclonal antibodies can be prepared by hybridoma methodologies. Monoclonal antibodies can also be isolated from phage antibody libraries using molecular engineering techniques. The monoclonal antibodies of this disclosure may be produced by recombinant DNA methods and may also be referred to as “recombinant antibodies” or “recombinant monoclonal antibodies” as described herein. In some embodiments, monoclonal antibodies are single-species antibodies in which all antibody molecules recognize the same epitope, since all antibody-producing cells originate from a single B lymphocyte cell line. Methods for producing monoclonal antibodies (mAbs) generally begin in much the same way as methods for preparing polyclonal antibodies. In some embodiments, rodents such as mice and rats are used to produce monoclonal antibodies. In certain embodiments, rabbit, sheep, or frog cells are used to produce monoclonal antibodies. The use of rats is well known and may offer certain advantages. Mice, for example, BALB / c mice, are routinely used and generally give a high percentage of stable fusion. In some embodiments of this disclosure, the antibody is a monoclonal antibody. In certain embodiments of this disclosure, the IgG antibody is monoclonal.

[0046] In some embodiments, recombinant antibody fragments can be isolated from phage antibody libraries using techniques well known in the art. See, for example, Clackson et al., 1991, Nature 352:624-628 and Marks et al., 1991, J.Mol.Biol.222:581-597. Recombinant antibody fragments may originate from large phage antibody libraries produced by recombination in bacteria (as described in Sblattero and Bradbury, 2000, Nature Biotechnology 18:75-80 and herein). The VH and VL components of the antibody fragment, i.e., the polynucleotides encoding scFv, can be used to generate recombinant full-length immunoglobulins using methods known in the art (see, for example, Persic et al., 1997, Gene 187:9-18).

[0047] "Isolated antibodies" are antibodies that have been identified, isolated, and / or recovered from the components of their existing environment. Contaminating components are materials that interfere with the therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.

[0048] As used herein, “human antibody” refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human. Human antibodies can be produced using various techniques known in the art, including phage display libraries. Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such an antibody in response to an antigen challenge, but whose endogenous locus has been deactivated. A “humanized” form of a non-human antibody, such as a rodent antibody, is a chimeric antibody containing the smallest sequence derived from a non-human antibody. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the hypervariable region of the recipient are replaced by residues from the hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired antibody specificity, affinity, and capabilities. In some embodiments, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antibody may contain residues not found in the recipient antibody or donor antibody. These modifications are made to further improve antibody performance. Generally, humanized antibodies contain substantially all of at least one, typically two, variable domains, all or substantially all of the hypervariable loops correspond to those of non-human immunoglobulins, and all or substantially all of the FRs are human immunoglobulin sequences. Humanized antibodies may also optionally contain immunoglobulin constant regions (Fc), typically at least a portion of the immunoglobulin constant regions (Fc) of human immunoglobulins.

[0049] Affinity-matured antibodies are those that have one or more changes in one or more hypervariable regions, resulting in improved antibody affinity to an antigen compared to parent antibodies that do not have those changes. In some embodiments, affinity-matured antibodies can have micromolar affinity to a target antigen. In some embodiments, affinity-matured antibodies can have nanomolar or even picomolar affinity to a target antigen. Affinity-matured antibodies are produced by procedures known in the art.

[0050] A “blocking” antibody or “antagonist” antibody inhibits or reduces the biological activity of the antigen to which it binds. In some embodiments, a blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of the antigen. As used herein, an “agonist antibody” is an antibody that mimics at least one of the functional activities of the polypeptide of interest. As used herein, terms such as “inhibit,” “inhibit,” and “inhibit” in relation to protein-inhibitor, e.g., antagonist interactions mean that the inhibitor adversely affects, for example, the activity or function of a protein in the absence of the inhibitor, for example, reduces it. In some embodiments, inhibition refers to the reduction of a disease or the symptoms of a disease. In some embodiments, inhibition refers to the reduction of activity in a signal transduction pathway or signaling pathway. Thus, inhibition includes, at least partially, partially or completely blocking a stimulus, reducing, preventing or delaying activation, or inactivating, desensitizing, or downregulating signaling or enzyme activity or the amount of a protein.

[0051] "Binding affinity" generally refers to the combined strength of non-covalent interactions between a molecule, e.g., a single binding site of an antibody, and its binding partner, e.g., an antigen. Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of a binding pair, e.g., an antibody and an antigen. The affinity of molecule X for its partner Y can generally be expressed by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally tend to bind slowly to antigens and dissociate easily, while high-affinity antibodies generally tend to bind more quickly to antigens and remain bound for longer. Various methods for measuring binding affinity are known in the art, and any of them can be used for the purposes of this disclosure. "Epitope" generally refers to the portion of an antigen to which it is bound by the antigen-binding site of an antibody. In some embodiments, the epitope may be "linear" in the sense that the hypervariable loop of the antibody CDR that forms the antigen-binding site binds to an amino acid sequence as it is in the primary protein structure. In some embodiments, the epitope is a "conformational epitope," i.e., the hypervariable loop of the CDR binds to residues that they present in the tertiary or quaternary protein structure.

[0052] In some embodiments of the compositions described above, the therapeutic biological preparation is a hormone. In some embodiments, the hormone includes, but is not limited to, testosterone (e.g., testosterone enanthate), dihydrotestosterone, androstenedione, estrone, estradiol, estriol, estrol, estrogen, and / or progesterone. In some embodiments, the therapeutic biological preparation (e.g., hormone) is dissolved in a non-aqueous vehicle (e.g., a carrier liquid, oil) containing the particles of the Disclosure. In some embodiments, the therapeutic biological preparation is XYOSTED® (e.g., testosterone enanthate).

[0053] In some embodiments of the compositions described above, the protein, for example, the therapeutic biological agent, is an antibody. In some embodiments, the antibody is not limited to, but may include 3F8, avagovomab, abatacept, absiximab, abituzumab, abrazekimab, abrilumab, acritumomab, actoxumab, abituzumab, adalimumab-adbm, adalimumab-atto, adalimumab-bwwb, adecatumumab, Ado-trastuzumab emtansine, aducanumab, afasebikumab, aferimomab, aflibercept, aftuzumab, aracizumab pegol, ALD518, alefacept, alemtuzumab, and aliloc. Mab, pentetate arthumomab, amatsuximab, anatumomab mafenatox, anddecaliximab, anetumab labutancin, aniflorumab, anlukinzumab, ansuvimab, apolizumab, apultumab ixadotin, arthumomab, ascurizumab, ascurizumab, atezolizumab, atidolotoxumab, atinumab, atorutivimab, atorumumab, atrolimumab, avelumab, adintuxizumab vedotin, bapineuzumab, basiliximab, bavituximab, BCD-100, vectumomab, vege Lomab, verantamab mahodotin, veratacept, belimumab, bemarituzumab, benralizumab, bermekimab, belsanlimab, vertilimumab, becilesomab, bevacizumab, bevacizumab-awwb, bezlotokisumab, bisilomab, bimaglumab, bimekizumab, vilutamimab, vivacuzumab meltansine, preserumab, blinatumomab, bronzvetomab, brosozumab, vococizumab, brazicumab, brentuximab vedotin, briakinumab, brodalumab, brolucizumab, bronticutuzumab, bros Mab, kabilizumab, camidanrumab tesirin, camrelizumab, canakinumab, cantuzumab meltansine, cantuzumab brabutansine, caplacizumab, capromab pendetide, carrumab, carotuximab, catumakisomab, cBR96-doxorubicin immune complex, sedelizumab, semiprimab, cergutuzumab amnaloykin, cergutuzumab amnaloykin, certolizumab pegol, cetrelimab, cetuximab, sibisatamab, silimutuzumab, sitatuzumab bogatox, sixtumumab, crazakizumab,Clenoliximab, cribatuzumab tetraxetan, codolituzumab, cofetuzumab peridotin, coltuximab labutansine, conatumumab, concizumab, cosflobiximab, crenezumab, CR6261, chryzanlizumab, clotezumab, kusatuzumab, dasetuzumab, dacrizumab, darotuzumab, dapirolizumab pegol, daratumumab, dectrekumab, demcizumab, denileukin diffittox, denintuzumab mahodotin, denosumab, depatuxizumab mahodotin, delrotuximab biotin, detumomab, dezamizumab, dji Nutuximab, ziridabumab, domaglozumab, dorulimomab aritox, dostallimab, dorodizumab, DS-8201, durigotumab, dupilumab, durvalumab, dusigitumab, duvoltuxizumab, eclomeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab, erderumab, erezanumab, elgemzumab, elotuzumab, elcilimomab, emuctuzumab, emibetuzumab, emicizumab, enapotamab vedotin, enabatuzumab, enfortumab vedotin, enlimomab pegor, enov Rituzumab, enokizumab, enoticumab, encituximab, epitumomab citucetan, epoetin-alpha, epoetin-alpha-epbx, epratuzumab, eptinezumab, erulizumab, erzumaxomab, etanercept, etanercept-szzs, etalacizumab, etigirimab, etrolizumab, evinacumab, evolocumab, exevivirumab, factor VIII Fc fusion protein, factor IX Fc fusion protein, fanolesomab, falalimomab, falisimab, farletuzumab, facinumab, felbi Zumab, Fezakinumab, Fivatuzumab, Ficratuzumab, Figitumumab, Filgrastim, Filgrastim-sndz, Filibumab, Flambotumab, Fleticumab, Flotetuzumab, Hontolizumab, Horamab, Horabirumab, Fremanezumab, Fresolimmab, Flobosimab, Flunebetomab, Fluranumab, Futuximab, Galcanezumab, Galiximab, Ganituzumab, Gantenerumab, Gatipotuzumab, Gabirimomab, Gezibumab, Gemtuzumab-ozogamicin, Gebokizumab, Gilvetomab, Gymcirumab, Gilentuximab,Glenbatumumab vedotin, golimumab, gomiliximab, goslanemab, guselkumab, ibalizumab, IBI308, ibritumomab tiuxetan, iclucumab, idarucizumab, ifabotuzumab, igobomab, iradatuzumab vedotin, IMAB362, imarumab, imaprelimab, imusilomab, imugatuzumab, incrakumab, indatuximab tansine, indusatumab vedotin, inebilizumab, infliximab, infliximab-abda, infliximab-dyyb, infliximab-qbtx, intetum Inolimomab, Inotuzumab ozogamisin, Ipilimumab, Iomab-B, Iratumumab, Isatuximab, Iscarimab, Istilatumab, Itorizumab, Ixekizumab, Keriximab, Labetuzumab, Lacnotuzumab, Radilatuzumab vedotin, Lambrolizumab, Lamparizumab, Lanadelmab, Landgrozumab, Laprituximab emtansine, Larcabiximab, Lebrikizumab, Remaresomab, Rendarizumab, Lembervimab, Rendilumab, Reldelimumab, Leronlimab, Resofabumab, Retrizumab, Lexatumumab, Rivivirumab, Rifastuzumab Vedotin, Rigerizumab, Roncastuzumab Tecilin, Rosatuzumab Vedotin, Rilotomab Satetraxetan, Lintuzumab, Lirirumab, Rodelcizumab, Lokivetomab, Rorbotuzumab Meltansine, Lucatumumab, Rulizumab Pegol, Lumiliximab, Lumuletuzumab, Rupalzumab Amadotin, Lutikizumab, Maftivimab, Mapatumumab, Margetuximab, Marstacimab, Masurimomab, Mabrilimumab, Matsuzumab, Mepolizumab, Meterimumab, Miratuzumab, Minretumomab, Mirikiz Mab, milbetuximab sorabtansine, mitumomab, modotuximab, mogamulizumab, monalizumab, morolimumab, mosnetuzumab, motabizumab, moxetumomab pasdotox, muromonab-CD3, nacolomab butafenatox, namilumab, naptumomab estafenatox, naratuximab emtansine, narunatumab, natalizumab, nabixixizumab, nabibumab, naxitamab, nevacumab, necitumumab, nemolizumab, NEOD001, nererimomab, nesbakumab, netakimab, nimotuzumab, nirsevimab, nivolumab,Nofetumomab merpentan, obiltoxaximab, obinutuzumab, okalatuzumab, ocrelizumab, odesibimab, odesibimab-ebgn, ozurimomab, ofatumumab, oraratuzumab, oleculumab, orendalizumab, orokizumab, omalizumab, omblutamab, OMS721, onartuzumab, ontuxizumab, onbachirimab, opicinumab, oporutuzumab monatox, olegobomab, orticumab, oterixizumab, ochirimab, otreltuzumab, oxerumab, ozanezumab, ozoralizumab, pagibaximab, pa Ribizumab, pamlebrumab, panitumumab, pancomab, panobacumab, pulsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patrizumab, PDR001, pegfilgrastim-jmdb, pembrolizumab, pemtumomab, perakizumab, pertuzumab, paxerizumab, pizilizumab, pinatuzumab vedotin, pintumomab, pracumab, prozarizumab, pogalizumab, polatuzumab vedotin, ponezumab, polgabiximab, pracinezumab, prezarizumab, priliximab, pritoxaximab, pritumumab, P RO140, kirizumab, lacosumomab, radrezumab, rafibirumab, larpancizumab, ramucirumab, lanebetomab, ranibizumab, laxibakumab, labagalimab, ravulizumab, refanezumab, regavirumab, relatrimab, resumolumab, reslizumab, rilonacept, rilotumumab, linucumab, risankizumab, rituximab, rituximab-abbs, rituximab-arrx, rituximab-pvvr, ribabazumab pegor, ribabazumab pegor, lobatumumab, Rmab, lorezumab, romirukimab, romiplostim, romomo Sozumab, lontalizumab, rosmantuzumab, lovalpituzumab tesirin, lovalpituzumab tesirin, loberizumab, rozanolixizumab, luprizumab, sacituzumab govitecan, samarizumab, samlotamab vedotin, saperizumab, sarilumab, satralizumab (SA237), satumomab pendetide, secukinumab, sericrelumab, cerivantuzumab, cetoxaximab, cetorusumab, sevilumab, cibrotuzumab, SGN-CD19A, SGN-CD33A, SHP647, cifalimumab, siltuximab, simtuzumab, ciprizumab,Siltratumab vedotin, siltratumab, sofituzumab vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, spartalizumab, stamlumab, thresomab, subutabumab, stimulimab, subizumab, subbratokisumab, tabarumab, tacutuzumab tetraxetan, tadocizumab, tafacitamab, talakotsuzumab, talizumab, tamtuvetomab, tanezumab, tapritumomab paptokis, tarekisuzumab, tavolimab, tefivazumab, terimomab aritokis S, terisotuzumab vedotin, tenatumomab, teneriximab, teprizumab, tepositamab, teprotumumab, tesidorumab, tetulomab, tezeperumab, TGN1412, tibrizumab, tisilimmab, tildrakizumab, tigatuzumab, timigutuzumab, timorumab, tyragotuzumab, tislerizumab, tisotuzumab vedotin, TNX-650, tocilizumab, tomzotuximab, tralizumab, tosatokisumab, tositumomab, tobetuzumab, tralokinumab, trastuzumab, Trastuzumab-ANNS, Trastuzumab-DKST, Trastuzumab-DTTB, Trastuzumab Emtansine, Trastuzumab-PKRB, Tregalizumab, Tremelimumab, Trevoglumab, Tucothuzumab Cermoloukin, Tubilumab, Ublituximab, Urocpurumab, Urelumab, Urtoxazumab, Ustekinumab, Utomirumab, Vadastuzumab Butarilin, Banarimab, Bundutuzumab Vedotin, Vanucizumab, Vanucizumab, Bapariximab, Barisac This includes mab, valrirumab, baterizumab, vedolizumab, beltuzumab, bepalimomab, besenkumab, vizilizumab, bovalilizumab, boroxiximab, bonrelorizumab, voplaterimab, borsetuzumab mahodotin, botumumab, xentuzumab, XMAB-5574, zaltumumab, zanolimmab, zatuximab, xenoctuzumab, diralimumab, zolbetuximab (IMAB362, claudicimab), Ziv-aflibercept, or zolimomab aritox.

[0054] In some embodiments of the compositions described above, the antibody is monoclonal. In certain embodiments, the monoclonal antibody may include, but is not limited to, 3F8, 8H9, abatacept, avagovomab, absiximab, abituzumab, adalimumab-adbm, adalimumab-atto, adalimumab-bwwb, abrilumab, actoxumab, abituzumab, abrazekimab, abrilumab, actoxumab, adalimumab, adecatumumab, Ado-trastuzumab emtansine, aducanumab, afasebicumab, aferimomab, aflibercept, aftuzumab, aracizumab pegol, ALD518. Alefacept, alemtuzumab, alirocumab, pentetate artumomab, amatsuximab, anatumomab mafenatox, anddecaliximab, anetumablavtansine, aniflorumab, anlukinzumab (IMA-638), ansuvimab, apolizumab, alsitumomab, askrinbakumab, aselizumab, atezolizumab, atidolotokisumab, atinumab, atorizumab (tocilizumab), atorutivimab, atrolimumab, avelumab, bapineuzumab, basiliximab, bevacizumab, bevacizumab-awwb BCD-100, vectumomab, begeromab, veratacept, belimumab, bemarituzumab, benralizumab, bermekimab, bersanlimab, bertilimumab, besilesomab, bezlotokisumab, bisilomab, bimaglumab, bimekizumab, vilutamimab, vibatuzumab meltansine, preserumab, blinatumomab, bronzvetomab, brosozumab, vococizumab, brazicumab, brentuximab vedotin, briakinumab, brodalumab, brolucizumab, bronticutuzumab, brosumab, kabilalizumab, camrelizumab Canakinumab, Cantuzumab Meltansine, Cantuzumab Brabutansine, Caplacizumab, Capromab Pendetide, Carrumab, Carotuximab, Katsumakisomab, Sedelizumab, Semiprimab, Certolizumab Pegol, Cetrelimab, Cetuximab, Sibisatamab, Silumutuzumab, Ch.14.18, Sitatuzumab Bogatox, Sixtumumab, Clazakizumab, Clenoliximab, Cribatuzumab Tetraxetan, Codlituzumab, Cofetuzumab Peridotin, Coltuximab Brabutansine, Conatumumab, Concizumab,Cosflobiximab, crenezumab, CR6261, chryzanlizumab, clotezumab, kusatuzumab, dasetuzumab, dacrizumab, dalotuzumab, dapirolizumab pegol, daratumumab, dectrekumab, demcizumab, denileukin diffittox, denintuzumab mahodotin, denosumab, delrotuximab biotin, detumomab, dezamizumab, dinutuximab, ziridabumab, domaglozumab, dorulimomab aritox, dostalurimab, dorozizumab, durigotuzumab, dupilumab, durvalumab, dusigituzumab, duvolutxy Zumab, eclomeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab, erderumab, erezanumab, elgemzumab, elotuzumab, elcilimomab, emuctuzumab, emibetuzumab, emicizumab, enabatuzumab, enfortumab vedotin, enrimomab pegol, enobrituzumab, enokizumab, enoticumab, encituximab, epitumomab citucetan, epoetin-alpha, epoetin-alpha-epbx, epratuzumab, eptinezumab, erenumab, erlizumab, ertzumaxomab Etanercept, etanercept-szzs, etalacizumab, etigirimab, etrolizumab, evinacumab, evolocumab, exevivirumab, factor VIII Fc fusion protein, factor IX Fc fusion protein, phanolesomab, falalimomab, falisimab, farletuzumab, facinumab, FBTA05, felbizumab, fezakinumab, fibatuzumab, ficratuzumab, figitumumab, filgrastim, filgrastim-sndz, firivumab, frambotumab, fretizumab, fluotetuzumab, hontolizumab, ho Larumab, horavirmab, fremanezumab, fresolimmab, flubocimab, flunebetomab, fluranumab, futuximab, galcanezumab, galiximab, ganitumab, gantenerumab, gatipotuzumab, gabirimomab, gezibumab, gemtuzumab ozogamicin, gebokizumab, gilvetomab, gymcirumab, gilentuximab, glenbatumumab vedotin, golimumab, gomiliximab, gosuranemab, guselkumab, ibalizumab, IBI308, ibritumomab tiuxetan, iclucumab, idarucizumab, ifabotuzumab, igobomab,IMAB362, Imarumab, Imaprelimab, Imsilomab, Imugatuzumab, Inlacrumab, Indatuximab tansine, Indusatuzumab vedotin, Innebilizumab, Infliximab, Infliximab-ABDA, Infliximab-DYYB, Infliximab-QBTX, Intetumumab, Inorimomab, Inotuzumab ozogamisin, Ipilimumab, Iratumumab, Isatuximab, Iscarimab, Istilatuzumab, Itorizumab, Ixekizumab, Keriximab, Labetuzumab, Lacunotuzumab, Lambrolizumab, Lamparizumab , lanadermab, landgrozumab, larcabiximab, lebrikizumab, remalesomab, lendarizumab, lembervimab, rendilumab, reldelimumab, leronlimab, resofabumab, letrizumab, lexatumumab, rivivirumab, rifastuzumab vedotin, rigerizumab, rilotomab satetraxetan, lintuzumab, lirirumab, rodelcizumab, lokivetomab, rorbotuzumab meltansine, lucatumumab, rulizumab pegol, lumiliximab, lumuretuzumab, rutikizumab, maftibimab, mapatumumab, margetuximab, ma Lustacimab, Masurimomab, Mabrilimumab, Matuzumab, Mepolizumab, Meterimumab, Miratuzumab, Minretumomab, Mirikizumab, Milbetuximab sorabtansine, Mitsumomab, Modotuximab, Mogamulizumab, Monalizumab, Morolimmab, Mosnetuzumab, Motabizumab, Moxetumomab Pasdotox, Muromonab-CD3, Nacolomabutafenatox, Namilumab, Naptumomab Estafenatox, Narunatumab, Natalizumab, Navixixizumab, Navibumab, Naxitamab, Nevacumab, Necitumumab, Nemolizumab, NE OD001, nererimomab, nesbakumab, netakimab, nimotuzumab, nircevimab, nivolumab, nofetumomab merpentan, obiltoxaximab, obinutuzumab, okalatuzumab, ocrelizumab, odesibimab, odesibimab-ebgn, ozurimomab, ofatumumab, oraratumab, olekurumab, orendalizumab, orokizumab, omalizumab, omblutamab, OMS721, onartuzumab, ontuxizumab, onbachirimab, opicinumab, oporutuzumab monatox, olegobomab, orticumab, otelixizumab,Otili-mab, Otreltuzumab, Oxerumab, Ozanezumab, Pagibaximab, Palivizumab, Pamrebulumab, Panitumumab, Pancomab, Panobacumab, Pulsatuzumab, Pascolizumab, Pasotuxizumab, Pateclizumab, Patrizumab, PDR001, Pegfilgrastim-jmdb, Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Paxerizumab, Pizilizumab, Pinatuzumab Vedotin, Pintumomab, Prakurumab, Prozarizumab, Pogalizumab, Polatuzumab Vedotin, Ponezumab, Polgabiximab, Placi Nezumab, prezarizumab, priliximab, pritoxaximab, pritumumab, PRO140, kirizumab, tetulomab, lacosumomab, radrezumab, rafibirumab, larpancizumab, ramucirumab, lanebetomab, ranibizumab, laxibakumab, labagalimab, ravulizumab, refanezumab, regavirumab, relatrimab, resumolumab, reslizumab, rilonacept, rilotumumab, linucumab, risankizumab-rzaa, rituximab, rituximab-abbs, rituximab-arrx, rituximab-pvvr, lobatumumab Rmab, lorezumab, romilukimab, romiplostim, romosozumab, lontalizumab, rosmantuzumab, roberizumab, rozanolixizumab, luprizumab, sacituzumab govitecan, samarizumab, sarilumab, satralizumab (SA237), satumomab pendetide, secukinumab, sericrelumab, cerivantuzumab, cetoxaximab, cetorusumab, sevilumab, cibrotuzumab, SGN-CD19A, SGN-CD33A, SHP647, cifalimumab, siltuximab, simtuzumab, ciprizumab, silumab, sofituzumab Vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, spartalizumab, stamlumab, thresomab, subutabumab, stimulimab, subizumab, subbratokisumab, tavarumab, tacuzumab tetraxetan, tadocizumab, tafacitamab, tarakotsuzumab, talizumab, tamtuvetomab, tanezumab, tapritumomab paptokis, tarekisuzumab, tavolimab, tefivazumab, terimomab aritokis, tenatumomab, teneriximab, teprizumab, tepositamab, teprotumumab, tesidorumab, tetulomab (rirotomab),Tezeperumab, TGN1412, tibrizumab, tisilimmab (tremerimumab), tildrakizumab, tigatuzumab, timigutuzumab, thymorumab, tiragotuzumab, tislerizumab, TNX-650, tocilizumab (atlizumab), tomzotuximab, tralizumab, tosatokisumab, tositumomab, tobetuzumab, tralokinumab, Trastuzumab, Trastuzumab-ANNS, Trastuzumab-DKST, Trastuzumab-DTTB, Trastuzumab Emtansine, Trastuzumab-PKRB, TRBS07, Tregalizumab, Tremelimumab, Trevoglumab, Tucothumab Cermoloukin, Tubilumab, Ublituximab, Urocuprumab, Urelumab, Ultoxin This includes sazumab, ustekinumab, utomirumab, banarimab, bundletuzumab vedotin, vanchikutuzumab, vanucizumab, bapariximab, valisakumab, valrirumab, baterizumab, vedolizumab, bertuzumab, bepalimomab, besenkumab, vizilizumab, bovalilizumab, borosiximab, bonrelorizumab, voplaterimab, borsetuzumab mahodotin, botumumab, xentuzumab, XMAB-5574, zaltumumab, zanolimumab, zatuximab, xenoctuzumab, diralimumab, zolbetuximab (IMAB362, claudiximab), Ziv-aflibercept, zolimomab aritox, or the corresponding anti-drug antibodies in human patient samples. In certain embodiments, the monoclonal antibody is rituximab, rituximab-abbs, rituximab-arrx, or rituximab-pvvr. In some embodiments, the monoclonal antibody is atorutibimab, maftibimab, odesibimab-ebgn, or a combination thereof.

[0055] In some embodiments, the monoclonal antibody is a biosimilar. In some embodiments, the biosimilar includes, but is not limited to, adalimumab-adbm, adalimumab-atto, adalimumab-bwwb, bevacizumab-awwb, epoetin alfa-epbx, etanercept-szzs, infliximab-abda, infliximab-dyyb, infliximab-qbtx, filgrastim-sndz, odesibimab-ebgn, pegfilgrastim-jmdb, pegfilgrastim-bmez, risankizumab-rzaa, rituximab-abbs, rituximab-arrx, rituximab-pvvr, trastuzumab-anns, trastuzumab-dttb, trastuzumab-pkrb, or trastuzumab-dkst. In certain embodiments, the active biosimilar substance is adalimumab, bevacizumab, enoxaparin sodium, epoetin alfa, epoetin zeta, etanercept, filgrastim, folitropin alfa, infliximab, insulin glargine, insulin lispro, pegfilgrastim, risankizumab, rituximab, rituximab-abbs, rituximab-arrx, rituximab-pvvr, somatropin, teriparatide, trastuzumab, trastuzumab-anns, trastuzumab-dttb, trastuzumab-pkrb, or trastuzumab-dkst. In some embodiments, the biosimilar is rituximab, rituximab-abbs, rituximab-arrx, or rituximab-pvvr. In certain embodiments, the biosimilar is trastuzumab-anns, trastuzumab-dttb, trastuzumab-pkrb, or trastuzumab-dkst.

[0056] In some embodiments, the targeting portion is an antibody from an intact polyclonal antibody, an intact monoclonal antibody, an antibody fragment, a single-chain Fv(scFv) variant, a multispecific antibody, a bispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, a fusion protein containing the antigenic determinant portion of an antibody, or another modified immunoglobulin molecule containing an antigen recognition site.

[0057] In certain embodiments, therapeutic biological agents include ledipasvir / sofosbuvir, insulin glargine, lenalidomide, 13-valent pneumococcal conjugate vaccine, fluticasone / salmeterol, elvitegravir / cobicistat / emtricitabine / tenofovir alafenamide, emtricitabine, rilpivirine, and tenofovir alafenamide, emtricitabine / tenofovir alafenamide, grazoprevir / elbasvir, coagulation factor VIIa recombinant, epoetin alfa, aflibercept, or etanercept.

[0058] In some embodiments, the therapeutic biological agent is abatacept, abobotulinum toxin A, agalsidase beta, albiglutide, aldesleukin, alglucosidase alfa, alteplase (cathoflo activase), anakinra, asfotase alfa, asparaginase, asparaginase erwinia chrysanthemi, becaprelmin, belatacept, collagenase, collagenase clostridium histolyticum, darbepoetin alpha, denileukin diffitox, dolanase alpha, dulaglutide, ecalantide, efgultigimod alpha-fcab, erosulfase alpha, etanercept-szzs, filgrastim, filgrastim-sndz, galsulfase, glucarpidase, idursulfase, incobotulinum toxin A, interferon alpha-2b, interferon alpha-n3, interferon beta-1a, interferon beta-1b, interferon gamma-1b, laronidase, methoxypolyethylene glycol-epoeth These include beta-, metreleptin, ocliplasmin, onabotulinum toxin A, oprelbequin, palifermin, parathyroid hormone, pegaspargase, pegfilgrastim, peginterferon alpha-2a, peginterferon alpha-2b, peginterferon beta-1a packaged with ribavirin, pegroticase, rasburicase, leteplase, lilonacept, limabotulinum toxin B, romiplostim, salglamostim, seberipase alpha, Tbo-filgrastim, tenecteplase, or Ziv-aflibercept.

[0059] Proteins, for example, therapeutic biological agents in particles disclosed herein, have an activity of about 0.5 to about 1.0 per unit, an activity of about 0.75 to about 1.0 per unit, or an activity of about 0.9 to about 1.0 per unit. The activity is measured for the same protein before particle formation. In some embodiments, the protein has an activity of about 0.5 to about 1.0 per unit. The term "activity" refers to the ratio of functional or structural aspects of a protein, for example, an antibody, antibody fragment, bovine serum albumin (BSA), or human serum albumin (HSA), at two time points. The denominator of this ratio corresponds to a measure of the functional or structural aspect of the protein in the aqueous supply solution immediately before droplet formation. The numerator of this ratio corresponds to the same measure of the functional or structural aspect of the protein at a later time point, for example, immediately after particle formation.

[0060] In some embodiments, the compositions and methods disclosed herein further include administering a pharmaceutically effective amount of a hyaluronan degrading agent that can be used to enhance exposure to a therapeutic biological product. Subcutaneous tissue or extracellular matrix consists of a network of fibrous proteins embedded within a viscoelastic gel of glycosaminoglycans. Hyaluronan is a prominent glycosaminoglycan in subcutaneous tissue. Hyaluronan is secreted into the interstitium by fibroblasts as a viscous polymer, which is then degraded locally and in the lymph by the action of hyaluronidase. Glycosaminoglycans are complex linear polysaccharides of the extracellular matrix, characterized by repeating disaccharide structures of N-substituted hexosamines and uronic acids, as in the case of hyaluronan.

[0061] Particles containing at least one therapeutic biological agent and a hyaluronan degrader can enhance the subcutaneous administration of compositions containing multiple particles by increasing and / or increasing the volume of the composition, for example, by injection, thereby improving the absorption of the therapeutic biological agent. The use of a hyaluronan degrader in the particles, such as hyaluronidase, can improve the subcutaneous administration of therapeutic biological agents into the systemic circulation through reversible hydrolysis of hyaluronan, such as reversible degradation of hyaluronan. Degradation of hyaluronan in the extracellular matrix temporarily opens channels in the subcutaneous tissue, thereby allowing a larger volume to be safely and comfortably administered to the subcutaneous tissue. Furthermore, degradation of hyaluronan temporarily reduces the viscosity of the subcutaneous tissue, promoting the dispersion of the injected liquid and facilitating their absorption, thereby increasing the bioavailability of the therapeutic biological agent (see, for example, Example 3). The effect of hyaluronidase is local and reversible, accompanied by complete reconstruction of the hyaluronan tissue, which occurs within 24–48 hours. See, for example, Frost, GI, “Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration”, Expert Opinion on Drug Delivery, 2007;4:427-440. The increased subcutaneous tissue permeability due to hyaluronan degradation correlates with the effectiveness of hyaluronidases in increasing the dispersion and absorption of compositions containing multiple particles administered subcutaneously. In certain embodiments, the methods described herein further comprise administering a pharmaceutically effective composition containing multiple particles suspended in a pharmaceutically acceptable liquid carrier, substantially all of which comprise at least one therapeutic biological agent or a salt thereof and a hyaluronan degrader. In some embodiments, the particles comprise at least two therapeutic biological agents or salts thereof. In certain embodiments, the particles comprise at least two antibodies and a hyaluronan degrader.In some embodiments, the particles include at least two monoclonal antibodies and a hyaluronan degrading agent.

[0062] In certain embodiments, the hyaluronan degrader is a hyaluronidase enzyme, also referred to herein as hyaluronidase (e.g., human HYAL1, HYAL2, HYAL3, HYAL4, and HYAL5). In some embodiments, the hyaluronidase is a soluble neutral-active hyaluronidase. In certain embodiments, the hyaluronidase is a mammalian hyaluronidase. In certain embodiments, the hyaluronidase is a non-mammalian hyaluronidase. In certain embodiments, the mammalian hyaluronidase is a human hyaluronidase (e.g., a human hyaluronidase having a UniProtKB accession number selected from P38567, P38567-2, Q5D1J4). In certain embodiments, the human hyaluronidase is a recombinant human hyaluronidase, e.g., rHuPH20. Certain recombinant human hyaluronidases suitable for use in the compositions disclosed herein are commercially available, for example, rHuPH20 from Halozyme Therapeutics (San Diego, CA) or Creative Biomart (Shirley, NY). References to hyaluronidases herein include precursor (e.g., untreated) and mature (e.g., treated) hyaluronidase polypeptides (e.g., those with the signal sequence removed), variants encoded by their cleavage forms, allele variants, species variants, splice variants, and other variants, including polypeptides having at least 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to polypeptides having the following amino acid sequences:LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVID WEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTA LYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY (Sequence ID 1).

[0063] In some embodiments, the hyaluronidase enzyme is recombinant human hyaluronidase. In some embodiments, the hyaluronidase enzyme is TERGASE® (recombinant human hyaluronidase), ALT-BC4 (recombinant human hyaluronidase), ALT-B4 (recombinant human hyaluronidase), MK-5180 (recombinant human hyaluronidase), HYLENEX® (recombinant human hyaluronidase), AMPHADASE® (bovine testicular hyaluronidase), HYDASE® (trademark) (bovine testicular hyaluronidase), VITRASE® (trademark) (bovine testicular hyaluronidase), Vespula vulgaris hyaluronidase, Apis mellifera hyaluronidase, Dolichovespula maculata hyaluronidase, Polistes annularis hyaluronidase, Mus musculus hyaluronidase, Sus scrofa hyaluronidase, Rattus hyaluronidases that have an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of the aforementioned hyaluronidases, including norvegicus hyaluronidase, Oryctolagus cuniculus hyaluronidase, Pongo pygmaeus hyaluronidase, Macaca fascicularis hyaluronidase, Cavia porcellus hyaluronidase, Staphylococcus aureus hyaluronidase, Staphylococcus pyogenes hyaluronidase, Clostridium perfringens hyaluronidase, combinations thereof, or one or more of the aforementioned hyaluronidases.In some embodiments, hyaluronidase is human hyaluronidase, bovine hyaluronidase (e.g., bovine testicular hyaluronidase), sheep hyaluronidase (e.g., sheep testicular hyaluronidase), Vespula vulgaris hyaluronidase, Apis mellifera hyaluronidase, Dolichovespula maculate hyaluronidase, Polistes annularis hyaluronidase, Mus musculus hyaluronidase, Sus scrofa hyaluronidase, Rattus norvegicus hyaluronidase, Oryctolagus cuniculus hyaluronidase, Pongo pygmaeus hyaluronidase, Macaca fascicularis hyaluronidase, Cavia porcellus hyaluronidase, Staphylococcus This includes aureus hyaluronidase, Staphylococcus pyogenes hyaluronidase, Clostridium perfringens hyaluronidase, any of the aforementioned recombinant hyaluronidases, or a combination thereof.

[0064] According to certain embodiments of this disclosure, the amount of hyaluronan degrading agent in the particles is less than about 3% by weight, for example, about 2% by weight, 1% by weight, 0.9% by weight, 0.8% by weight, 0.7% by weight, 0.6% by weight, 0.5% by weight, 0.4% by weight, 0.3% by weight, 0.2% by weight, 0.1% by weight, 0.09% by weight, 0.08% by weight, 0.07% by weight, 0.06% by weight, 0.05% by weight, 0.04% by weight, 0.03% by weight, 0.02% by weight, or less than about 0.01% by weight. In some embodiments, the amount of hyaluronan degrading agent in the particles is less than about 0.1% by weight. In certain embodiments, the amount of hyaluronan degrading agent in the particles is less than about 0.5% by weight.

[0065] According to certain embodiments of this disclosure, the hyaluronan degrading agent in the particles may be less than about 100% by weight, for example, about 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50% by weight, 45% by weight, 40% by weight, 35% by weight, 30% by weight, 25% by weight, 20% by weight, 15% by weight, 10% by weight, 9% by weight, 8% by weight, 7% by weight, 6% by weight It is either 5% by weight, 4% by weight, 3% by weight, 2% by weight, 1% by weight, 0.9% by weight, 0.8% by weight, 0.7% by weight, 0.6% by weight, 0.5% by weight, 0.4% by weight, 0.3% by weight, 0.2% by weight, 0.1% by weight, 0.09% by weight, 0.08% by weight, 0.07% by weight, 0.06% by weight, 0.05% by weight, 0.04% by weight, 0.03% by weight, 0.02% by weight, or less than approximately 0.01% by weight.

[0066] A therapeutically effective single dose of a hyaluronane-degrading agent, such as hyaluronidase, is approximately 200 to 50,000 units, for example, approximately 600 to 6,000 units of hyaluronidase. For example, a hyaluronane-degrading agent, such as hyaluronidase, can be administered subcutaneously in doses of approximately 200, 500, 600, 1,000, 2,000, 6,000, 10,000, 16,000, less than 20,000 units, or less than approximately 50,000 units. In some embodiments, the hyaluronane-degrading agent, such as hyaluronidase, in suspension has a concentration of less than approximately 50,000 U / mL, for example, less than approximately 20,000, 16,000, 10,000, 6,000, 2,000, 1,000, 600, 500, or less than approximately 200 U / mL. In some embodiments, the hyaluronan degrading agent in the suspension has a concentration of less than about 6000 U / mL. In certain embodiments, the hyaluronan degrading agent in the suspension has a concentration of less than about 2000 U / mL. As used herein, the stability of the hyaluronan degrading agent, e.g., hyaluronidase, in particles and / or compositions means that it retains at least 50%, 60%, 70%, 80%, or 90% of the activity of an effective amount of hyaluronan degrading agent, e.g., hyaluronidase, before particle formation. In some embodiments, the stability of the hyaluronidase in particles and / or compositions is at least 70% of the activity of an effective amount of hyaluronidase before particle formation. In certain embodiments, the stability of the hyaluronidase in particles and / or compositions is at least 90% of the activity of an effective amount of hyaluronidase before particle formation.

[0067] As used herein, hyaluronidase activity, for example, effective amount, refers to the ability of hyaluronidase to cleave hyaluronic acid. In vitro assays for determining the hyaluronidase activity of hyaluronidases such as soluble rHuPH20 are known in the art and are described herein.

[0068] In some embodiments, the particles comprise at least one therapeutic biological agent and a hyaluronan degrader. In certain embodiments, the particles comprise at least two therapeutic biological agents and a hyaluronan degrader. In some embodiments, the particles comprise at least two antibodies and a hyaluronan degrader. In certain embodiments, the particles comprise at least two monoclonal antibodies and a hyaluronan degrader.

[0069] In some embodiments, substantially all of the particles have less than about 10% of the processed protein, e.g., less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% of the therapeutic biological product. In some embodiments, substantially all of the particles have less than about 5% of the therapeutic biological product. In certain embodiments, substantially all of the particles have less than about 3% of the therapeutic biological product. In some embodiments, substantially all of the particles have less than about 1% of the therapeutic biological product. In certain embodiments, substantially all of the particles contain substantially no aggregates of the therapeutic biological product. In some embodiments, substantially all of the particles have less than about 0.5% of the therapeutic biological product.

[0070] In some embodiments, substantially all of the particles have less than 10% fragmentation of the processed protein, e.g., the therapeutic biological product. In some embodiments, substantially all of the particles have less than 3% fragmentation of the therapeutic biological product. In certain embodiments, substantially all of the particles have less than 1% fragmentation of the therapeutic biological product. In some embodiments, substantially all of the particles contain substantially no fragmentation of the therapeutic biological product. In some embodiments, substantially all of the particles have less than 5% fragmentation of the therapeutic biological product. A preferred method for measuring the coagulation and fragmentation of the therapeutic biological product can be achieved by using size exclusion chromatography (SEC).

[0071] In certain embodiments, the composition provides a change of less than approximately 5% (e.g., less than approximately 4, 3, 2, or 1%) in charge variants in a population of therapeutic biological agents compared to the therapeutic biological agent before particle formation. In some embodiments, the particles have a change of less than approximately 5% in charge variants in a population of therapeutic biological agents after treatment. The charge variants may be acidic, basic, or neutral, and the variation may be caused by post-translational modifications at terminal amino acids, such as asparagine deamidation or lysine glycosylation. For example, charge variants include loss of positive charge due to loss of a C-terminal lysine residue, covalent bonding of the amine moieties of two lysine residues by a reducing sugar, or conversion of the N-terminal amine to a neutral amide by cyclization of the N-terminal glutamine. Negative charges on proteins, such as antibodies or fragments, may arise from the conversion of asparagine residues to aspartic and / or isoaspartic residues via deamidation reactions.

[0072] In some embodiments, substantially all particles have less than about 5% change in the charge variant of the protein, e.g., the therapeutic biological agent, compared to an aqueous composition containing at least one therapeutic biological agent in a soluble (monomer) form before particle formation. In some embodiments, substantially all particles have less than about 3% change in the charge variant of the therapeutic biological agent. In certain embodiments, substantially all particles have less than about 1% change in the charge variant of the therapeutic biological agent. In some embodiments, substantially all particles contain substantially no change in the charge variant of the therapeutic biological agent. An exemplary method for measuring the charge variant includes cation exchange chromatography (CIEX), where the variant is quantified by dividing the area under the peak corresponding to the variant, e.g., acidic, basic, or neutral population, by the cumulative area contained under all peaks in the sample spectrum. The change in the charge variant population percentage between two samples, for example, sample A and sample B, is calculated as a numerical difference in the respective population variant percentages by subtracting the percentage of a specific variant in sample B, e.g., the acidic variant, from the percentage of a specific variant in sample A, e.g., the acidic variant, or vice versa. In certain embodiments, the analysis can be similarly extended to all variants within the population.

[0073] The particles according to this disclosure are circular. Roundness can serve as an indicator of the particle's shape. The particles described herein may have a characteristic roundness, for example, a relative shape that is substantially circular. This characteristic describes and defines the particle's morphology based on its roundness. If a particle has a perfectly circular structure, the roundness is 1.0. The particles according to this disclosure have roundnesses of about 0.80 to about 1.00, 0.90 to about 1.00, 0.95 to about 1.00, 0.96 to about 1.00, 0.97 to about 1.00, 0.98 to about 1.00, or 0.99 to about 1.00. In some embodiments, substantially all of the particles have a roundness of about 0.85 to about 1.00. In some embodiments, substantially all of the particles have a roundness of about 0.90 to about 1.00. In certain embodiments, the substantially all roundness of the particles is approximately 0.95 to approximately 1.00. In certain embodiments, the substantially all roundness of the particles is approximately 0.98 to approximately 1.00. In certain embodiments, the substantially all roundness of the particles is approximately 1.00. The diameter and roundness of the particles can be determined by processing images observed under an electron microscope or a flow-type particle image analyzer. Roundness can also be determined by subjecting the particles to roundness measurement and averaging the obtained values. For example, roundness (circ) can be calculated using the following formula:

number

[0074] As used herein, the term “perimeter” refers to the boundary of a closed planar figure or the sum of all borders of a two-dimensional image. As used herein, the term “area” refers to the cross-sectional area of ​​a two-dimensional image of a particle. The roundness of a particle can also be described as the ratio of the particle’s minimum dimension to its maximum diameter. For a perfect circle, this ratio is 1. The roundness percentage can be calculated by multiplying the roundness by 100. Roundness can be calculated, for example, by measuring the aspect ratio using any software adapted to handle images, e.g., images obtained by microscopy, in particular by scanning electron microscopy (SEM) or transmission electron microscopy (TEM). In some embodiments, the method for measuring particle roundness involves image analysis of a scanning electron microscope image of a particle, where the mean roundness is calculated based on the cross-sectional shape of the particle projected onto the plane of the image. Such a roundness coefficient can be extended to identify the corresponding roundness.

[0075] In certain embodiments of this disclosure, the particles described herein have a smooth surface morphology, rather than being raised or wrinkled. Those skilled in the art of this disclosure can easily evaluate the surface morphology of the disclosed particles using routine standard techniques.

[0076] In certain embodiments, the particles have a diameter of about 0.1 to about 5000 μm, for example, about 0.1 to about 4000, 3000, 2000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or about 0.2 μm. In some embodiments, the particles have a diameter of about 1 to about 100 μm, for example, about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 to about 100 μm. In some embodiments, the particles have a diameter of about 4 to about 100 μm. In certain embodiments, the particles have a diameter of about 10 to about 100 μm. In certain embodiments, the particles have a diameter of about 20 to about 50 μm. Methods for measuring particle size and distribution include imaging flow cytometry, laser diffraction, and image analysis of scanning electron microscope images of the particles, where the mean spherical radius or diameter can be calculated based on the cross-sectional area of ​​the particle projected onto the plane of the image.

[0077] As used herein, the term “dispersion index” (DI) is a parameter that characterizes the degree of heterogeneity in the particle size distribution. The polydispersity index (PDI), “collective dispersibility,” or “span,” e.g., D10, D50, D90, can also refer to values ​​indicating the width of the particle size distribution. The particle size distribution is reported by the average particle size in μm, D10, D50, D90, and D10, where the values ​​represent the percentage of particles smaller than the indicated D number. For example, D10 particle size is the particle diameter where 10% of the mass consists of particles with a diameter less than this value; D50 particle size is the particle diameter where 50% of the mass consists of particles with a diameter less than this value; and D90 particle size is the particle diameter where 90% of the mass consists of particles with a diameter less than this value. The D10, D50, and D90 particle size distributions can be measured using a laser light scattering particle analyzer.

[0078] Particle diameter and dispersibility can also affect injectability, e.g., syringeability. It is often recommended that particles be at least 3 to 10 times smaller than the inner diameter of the needle. Even small fractions of the particle population larger than the inner diameter of the needle can clog the needle and waste the entire dose. In certain embodiments of the present disclosure described herein, high concentrations of therapeutic biological formulations in a composition are achieved by mixing particles of varying sizes.

[0079] As used herein, the terms “moisture” and “water” may be used interchangeably. In some embodiments, the substantially total moisture content of particles is less than about 10% by weight, for example, less than about 9, 8, 7, 6, 5, 4, 3, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1% by weight. In some embodiments, substantially all particles have less than about 7% by weight of moisture. In certain embodiments, substantially all particles have less than about 5% by weight of moisture. In certain embodiments, substantially all particles have less than about 3% by weight of moisture. In some embodiments, substantially all particles have less than about 1% by weight of moisture. Exemplary methods for measuring moisture content include chemical titration, e.g., Karl Fischer titration with oven. Various solvents containing water may also be measured using weight loss methods with thermal excitation. Exemplary methods include thermogravimetric analysis with infrared spectroscopy (TGA-IR) or gas chromatography-flame ionization detector / mass spectrometry (GC-FID / MS).

[0080] As used herein, the term "internal void space" or "internal void", in contrast to pores or porosity, refers to a space that does not communicate with the surface of a solid (e.g., a particle) and does not contribute to porosity or surface area. In some embodiments, substantially all of the particles have less than about 25% internal void space, for example less than about 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% internal void space. In some embodiments, substantially all of the particles are substantially free of any internal void space. A suitable method for determining internal void space can be accomplished by using focused ion beam scanning electron microscopy (FIB-SEM), which can be used to visualize "accessible" pores and "inaccessible" void spaces. Another method for determining internal void space can be achieved by gas displacement pycnometry, a common analytical technique that uses gas displacement methods to measure volume. An inert gas such as helium or nitrogen is used as the displacement medium. The true volume is the total volume minus the gas-accessible volume. Density is calculated by dividing the sample weight by the true volume. The sample is sealed in an instrument compartment of known volume, charged with a suitable inert gas, and then the gas is expanded into another precise internal volume. The pressures before and after expansion are measured and used to calculate the sample volume. Divide the sample weight by this volume to obtain the gas displacement density. Cross-sections of typical particles of the present disclosure show the absence of any internal void space, as demonstrated by FIB-SEM or by gas pycnometry using helium at a temperature of about 22°C, and typically have a standard deviation of about 0.0005 g / cm 3 and an average density of about 1.3 g / cm 3 For example, the internal void space can be calculated using the following formula: Internal void space = A v / A p wherein A v is the total area of the void space, and A pis the total surface area of ​​the particles. Typical particle cross-sections of the present disclosure can be obtained and characterized in many ways, for example, in International Application No. PCT / US2020 / 15957 (Publication No. WO2020 / 160323) and PCT / US2021 / 027755 (WO2021 / 212019), the contents of each of these are incorporated herein by reference in their entirety.

[0081] In some embodiments, substantially all particles have less than about 25% internal void space. In some embodiments, substantially all particles have less than about 10% internal void space. In certain embodiments, substantially all particles have less than about 5% internal void space. In certain embodiments, substantially all particles have less than about 3% internal void space. In some embodiments, substantially all particles have less than about 1% internal void space. In certain embodiments, substantially all particles contain substantially no internal void space.

[0082] In some embodiments, substantially all particles contain more than about 50% by weight of protein, for example, therapeutic biological agents, for example, more than about 55, 60, 65, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% by weight of therapeutic biological agents. In some embodiments, substantially all particles contain more than about 60% by weight of therapeutic biological agents. In some embodiments, substantially all particles contain more than about 65% by weight of therapeutic biological agents. In certain embodiments, substantially all particles contain more than about 70% by weight of therapeutic biological agents. In some embodiments, substantially all particles contain more than about 80% by weight of therapeutic biological agents. In certain embodiments, substantially all particles contain more than about 90% by weight of therapeutic biological agents. In certain embodiments, substantially all particles contain more than about 95% by weight of therapeutic biological agents. In certain embodiments, substantially all of the particles contain more than about 98% by weight of a therapeutic biological agent. In some embodiments, substantially all of the particles contain more than about 99% by weight of a therapeutic biological agent.

[0083] The term “excipient” refers to an additive to a particle formulation that may be useful in achieving a desired modification to the characteristics or morphology of the particles. Such modifications include, but are not limited to, physical stability, chemical stability, and therapeutic efficacy. Exemplary excipients include, but are not limited to, carbohydrates, pH adjusters, salts, chelating agents, surfactants, protein stabilizers, emulsifiers, preservatives, amino acids, antioxidants, parabens, bactericides, fungicides, preservatives, or combinations thereof. In some embodiments, substantially all of the particles further comprise one or more excipients. In some embodiments, substantially all of the particles further comprise carbohydrates, pH adjusters, salts, chelating agents, surfactants, protein stabilizers, emulsifiers, amino acids, antioxidants, or combinations thereof. In some embodiments, substantially all of the particles comprise at least carbohydrates and amino acids. In some embodiments, substantially all of the particles comprise at least surfactants and amino acids. In certain embodiments, substantially all of the particles comprise at least one amino acid.

[0084] In some embodiments, the carbohydrate may belong to the family of monosaccharides, disaccharides, oligosaccharides, or polysaccharides. In some embodiments, the carbohydrate is dextran, trehalose, sucrose, agarose, mannitol, lactose, sorbitol, maltose, starch, alginate, xanthan gum, galactomanin, agar, agarose, or a combination thereof. In certain embodiments, the carbohydrate is dextran, trehalose, sucrose, agarose, mannitol, lactose, sorbitol, maltose, or a combination thereof. In certain embodiments, the carbohydrate is trehalose, cyclodextrin, hydroxypropyl beta-cyclodextrin, sulfobutyl ether beta-cyclodextrin, methyl beta-cyclodextrin, or a combination thereof. In certain embodiments, the carbohydrate is trehalose, sucrose, sorbitol, or a combination thereof. In some embodiments, the carbohydrate is sucrose. Cyclodextrins are available in three different forms, α, β, and γ, based on the number of glucose monomers. The number of glucose monomers in α, β, and γ cyclodextrins can be 6, 7, or 8, respectively.

[0085] The terms “pH adjuster,” “buffer species,” or “buffer” are used interchangeably in the broadest sense herein. In some embodiments, substantially all of the particles contain a pH adjuster. In some embodiments, substantially all of the particles contain one or more pH adjusters. In certain embodiments, the pH adjuster is acetate, citrate, glutamate, glycinate, histidine, lactate, maleate, phosphate, succinate, tartrate, bicarbonate, aluminum hydroxide, phosphoric acid, hydrochloric acid, DL-lactic acid / glycolic acid, phosphorylethanolamine, tromethamine, imidazole, glycryglycine, monosodium glutamate, sodium hydroxide, potassium hydroxide, or a combination thereof. In certain embodiments, the pH adjuster is acetate, citrate, histidine, phosphate, succinate, hydrochloric acid, sodium hydroxide, or a combination thereof. In certain embodiments, the pH adjuster is histidine, hydrochloric acid, or a combination thereof. In certain embodiments, the pH adjuster is histidine HCl, succinate, phosphate, or a combination thereof. In some embodiments, the pH level of the compositions disclosed herein is about 5.0 to about 8.0. In some embodiments, the pH level of the compositions disclosed herein is about 5.5 to about 7.0. In certain embodiments, the pH level is about 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5. In certain embodiments, the pH levels are approximately 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0.

[0086] In some embodiments, the salt is sodium chloride, calcium chloride, potassium chloride, sodium hydroxide, stannous chloride, magnesium sulfate, sodium glucoheptonate, sodium pertechnetate, guanidine hydrochloride, potassium hydroxide, magnesium chloride, potassium nitrate, or a combination thereof. In certain embodiments, the salt is sodium chloride, sodium hydroxide, potassium nitrate, magnesium chloride, or a combination thereof. In some particular embodiments, the salt is sodium chloride.

[0087] In some embodiments, the chelating agent is disodium edetate, ethylenediaminetetraacetic acid, pentetic acid, or a combination thereof.

[0088] In some embodiments, the surfactants are, but are not limited to, (i) cationic surfactants such as cetyltrimethylammonium chloride, hexadecyltrimethylammonium chloride, benzalkonium chloride, benzethonium chloride, and dioctadecyldimethylammonium bromide; (ii) anionic surfactants such as magnesium stearate, sodium stearate, sodium dodecyl sulfate, sodium dioctyl sulfosuccinate, sodium myreth sulfate, perfluorooctanesulfonate, alkyl ether phosphate, and Aerosol-OT (sodium bis(2-ethylhexyl) sulfosuccinate); (iii) polysorbate, polyethylene glycol tert-octylphenyl ether, lecithin, alkylphenol ethoxylate, fatty alcohol ethoxylate (octaethylene glycol monododecyl ether, cocamidodiethanolamine, poloxamer, glycerol monostearate, fatty amine, and fatty acid esters of sorbitol (sorbitan monolaurate, Tween 80, Tween 20. Nonionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) adjacent to two hydrophilic chains of poloxamer (polyoxyethylene (poly(ethylene oxide))), such as nonionic surfactants such as poloxamer 188 and poloxamer 407, as well as zwitterionic surfactants such as cocamidopropyl hydroxysultaine and 3-[(3-collamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS).In some embodiments, the surfactant is polysorbate, magnesium stearate, sodium dodecyl sulfate, alkylphenol ethoxylate, glycerin, polyoxyethylated castor oil, doxate, sodium stearate, decyl glucoside, nonoxynol-9, cetyltrimethylammonium bromide, sodium bis(2-ethylhexyl)sulfosuccinate, lecithin, sorbitan ester, phosphatidylcholine, polyglycerol polyricinoleate, siloxane, cetyl polyethylene glycol / polypropylene glycol-10 / 1 dimethicone triglyceride, bis-polyethylene glycol / polypropylene glycol-14 / 14 dimethicone, bis-(glyceryl / lauryl)glyceryl lauryl dimethicone (and) caprylic / capric triglyceride, cetyl polyethylene glycol / polypropylene glycol-10 / 1 dimethicone, phospholipids, or a combination thereof. In certain embodiments, the surfactant is polysorbate, doxate, poloxamer, or lecithin. In some embodiments, the surfactant is polysorbate 20, polysorbate 60, or polysorbate 80. In certain embodiments, the surfactant is polysorbate 20 or polysorbate 80, e.g., Tween 20, Tween 60, Tween 80. In certain embodiments, the fatty acid ester of sorbitol is sorbitan ester, e.g., span 20, span 40, span 60, or span 80. In some embodiments, the surfactant is an ionic surfactant. In certain embodiments, the surfactant is polysorbate 80, polysorbate 20, or poloxamer 188. In some embodiments, the surfactant is polysorbate 80. In some embodiments, the surfactant is a sorbitan ester, polysorbate, poloxamer, polyethylene glycol tert-octylphenyl ether, lecithin, sodium stearate, magnesium stearate, or a combination thereof.

[0089] In some embodiments, the protein stabilizer is acetyltryptophanate, caprylate, N-acetyltryptophan, trehalose, polyethylene glycol (PEG), polyoxomer, polyvinylpyrrolidone, polyacrylic acid, poly(vinyl)polymer, polyester, polyaldehyde, tert-polymer, polyamino acid, hydroxyethyl starch, N-methyl-2-pyrrolidone, sorbitol, sucrose, mannitol, or a combination thereof. In some embodiments, the protein stabilizer is trehalose, polyethylene glycol (PEG), polyoxomer, polyvinylpyrrolidone, polyacrylic acid, poly(vinyl) polymer, polyester, polyaldehyde, tert-polymer, polyamino acid, hydroxyethyl starch, N-methyl-2-pyrrolidone, sorbitol, sucrose, mannitol, cyclodextrin, hydroxypropyl beta-cyclodextrin, sulfobutyl ether beta-cyclodextrin, methyl beta-cyclodextrin, acetyltryptophanate, caprylate, N-acetyltryptophan, propylene glycol, glucose star polymer, silicone polymer, polydimethylsiloxane, carboxymethylcellulose, poly(glycolic acid), poly(lactic acid-coglycolic acid), polylactic acid, polycaprolactone (PCL), polyvinylpyrrolidone (PVP), Ficol, dextran, or a combination thereof. In certain embodiments, the protein stabilizer is trehalose, cyclodextrin, hydroxypropyl beta-cyclodextrin, sulfobutyl ether beta-cyclodextrin, methyl beta-cyclodextrin, or a combination thereof. In some embodiments, the protein stabilizer is hydroxypropyl beta-cyclodextrin, methyl beta-cyclodextrin, or a combination thereof. Stabilizers used herein in a manner synonymous with the term “stabilizer” may be salts, carbohydrates, sugars, or amino acids, preferably carbohydrates or sugars approved by the authorities as suitable additives or excipients in pharmaceutical compositions.In certain embodiments, PEG is PEG 200, PEG 300, PEG 3350, PEG 8000, PEG 10000, PEG 20000, or a combination thereof. The term “stabilizer” refers to an excipient or mixture of excipients that stabilizes the physical and / or chemical properties of a protein, such as an antibody or antibody fragment. In some embodiments, the stabilizer prevents, for example, the degradation of the protein during mixing and / or storage of particulate matter. Exemplary stabilizers include, but are not limited to, sugars, salts, hydrophobic salts, detergents, reducing agents, cyclodextrins, polyols, carboxylic acids, and amino acids. The “stable” formulation or composition described herein refers to a particulate formulation or composition comprising particles that retain an acceptable portion of the essential physical, chemical, or biological properties of a protein over an acceptable period of time. For proteins, exemplary methods for evaluating stability are reviewed in (i) Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, 1991, and (ii) Jones, A., Adv. Drug Delivery Rev. 10: 29-90 (1993). In some embodiments, the chemical stability of a protein is evaluated by measuring the size distribution of the sample at several stages. These include, for example, before particle formation (evaluation of the aqueous supply solution), immediately after particle formation, and again after the storage period, which is carried out either in or in the absence of a suspension carrier medium.

[0090] Examples of emulsifiers suitable for use in particles include, but are not limited to, lipophilic agents having an HLB of less than 7, e.g., mixed fatty acid monoglycerides; mixed fatty acid diglycerides; mixtures of fatty acid mono and diglycerides; lipophilic polyglycerol esters; glycerol esters including glyceryl monooleate, glyceryl dioleate, glyceryl monostearate, glyceryl distearate, glyceryl monopalmitate, and glyceryl dipalmitate; glyceryl-lacto esters of fatty acids; propylene glycol esters including propylene glycol monopalmitate, propylene glycol monostearate, and propylene glycol monooleate; sorbitan esters including sorbitan monostearate and sorbitan sesquioleate. Fatty acids including stearic acid, palmitic acid, and oleic acid, and soaps thereof; and mixtures thereof, including glyceryl monooleate, glyceryl dioleate, glyceryl monostearate, glyceryl distearate, glyceryl monopalmitate, and glyceryl dipalmitate; glyceryl-lacto esters of fatty acids; propylene glycol esters including propylene glycol monopalmitate, propylene glycol monostearate, and propylene glycol monooleate; sorbitan esters including sorbitan monostearate and sorbitan sesquioleate; fatty acids including stearic acid, palmitic acid, and oleic acid, and soaps thereof; phospholipids; or combinations thereof. In some embodiments, the emulsifier is polysorbate (polysorbate 80, polysorbate 60, polysorbate 20, e.g., Tween 80, Tween 60, Tween 20), sorbitan monooleate, ethanolamine, polyoxyl 35 castor oil, poloxyl 40 hydrogenated castor oil, carbomer 1342, corn oil mono-di-triglyceride, polyoxyethylated oleiceride, poloxamer, or a combination thereof. In some embodiments, the fatty acid ester of sorbitol is sorbitan ester, e.g., span 20, span 40, span 60, or span 80.In certain embodiments, the emulsifier is polysorbate 80, sorbitan monooleate, or a combination thereof.

[0091] In some embodiments, the preservative is phenol, m-cresol, benzyl alcohol, 2-phenyloxyethanol, chlorobutanol, neomycin, benzethonium chloride, gluteraldehyde, beta-propiolactone, or a combination thereof.

[0092] In certain embodiments, the amino acids are alanine, aspartic acid, cysteine, isoleucine, glutamic acid, leucine, methionine, phenylalanine, pyrrolicin, serine, selenocysteine, threonine, tryptophan, tyrosine, valine, asparagine, arginine, histidine, glycine, glutamine, proline, or various salts thereof (such as arginine hydrochloride and glutamic acid arginine), or combinations thereof. In some embodiments, the amino acids are alanine, aspartic acid, cysteine, isoleucine, glutamic acid, leucine, methionine, phenylalanine, pyrrolicin, serine, selenocysteine, threonine, tryptophan, tyrosine, valine, asparagine, arginine, histidine, glycine, glutamine, proline, or combinations thereof. In some embodiments, the amino acids are arginine, histidine, proline, asparagine, or combinations thereof. In certain embodiments, the amino acid is histidine. In some embodiments, the amino acid is alanine, glutamic acid, methionine, threonine, tryptophan, asparagine, arginine, histidine, glycine, glutamine, proline, or a combination thereof. In some embodiments, the amino acids are arginine and histidine. In certain embodiments, the amino acid is histidine.

[0093] In some embodiments, the antioxidant is glutathione, ascorbic acid, cysteine, N-acety-L-tryptophanate, tocopherol, histidine, methionine, pentetic acid, or a combination thereof. In some embodiments, the paraben is parahydroxybenzoate. In certain embodiments, the fungicide is benzalkonium chloride (a cationic surfactant), hypochlorite, peroxide, alcohol, phenolic compounds (e.g., carbolic acid), benzyl benzoate, or a combination thereof. In certain embodiments, the fungicide is benzalkonium chloride or benzyl benzoate. In certain embodiments, the fungicide is acibenzoral, 2-phenylphenol, anilazine, carvone, natamycin, potassium azide, or a combination thereof.

[0094] In some embodiments, the preservative is sodium nitrate, sulfur dioxide, potassium sorbate, sodium sorbate, sodium benzoate, benzoic acid, methyl hydroxybenzoate, thimerosal, parabens, formaldehyde, castor oil, or a combination thereof. In certain embodiments, the preservative is methyl hydroxybenzoate, thimerosal, parabens, formaldehyde, castor oil, or a combination thereof.

[0095] In some embodiments, insoluble particulate matter having a characteristic size of about 100 μm or larger that persists when dissolved in an aqueous liquid is referred to as visible particles (VP). In certain embodiments of the disclosure described herein, compositions, e.g., particles and compositions, are substantially free of visible particles (VP). In some embodiments, the aqueous liquid is water, an aqueous buffer, or a physiologically relevant aqueous liquid. As used herein, the term “physiologically relevant aqueous liquid” refers to any water-containing body fluid distributed in the extracellular compartment, e.g., extracellular fluid, interstitial fluid, intravascular fluid (blood, plasma, and lymph), and cerebrospinal fluid. In some embodiments, particles or compositions, when dissolved in an aqueous liquid, are substantially free of visible particles (VP).

[0096] In some embodiments, insoluble particulate matter having a characteristic size of about 1 μm to about 100 μm that persists when dissolved in an aqueous liquid is referred to as sub-visible particles (SvP). SvP exists in amounts of approximately 0 to 100,000,000 per mL, for example, approximately 0 to 10,000,000 per mL, approximately 0 to 1,000,000 per mL, approximately 0 to 500,000 per mL, approximately 0 to 100,000 per mL, approximately 0 to 50,000 per mL, approximately 0 to 10,000 per mL, approximately 0 to 6,000 per mL, approximately 0 to 1,000 per mL, approximately 0 to 600 per mL, approximately 0 to 250 per mL, approximately 0 to 100 per mL, approximately 0 to 60 per mL, or approximately 0 to 10 per mL. In some embodiments, the count of particles having a characteristic size of 10 μm or larger is about 0 to about 6,000 per mL, for example, about 0 to about 1,000, about 0 to about 100, about 0 to about 10, about 0 to about 5, about 0 to about 3, or about 0 to about 1 per mL. In a particular embodiment, the count of particles having a characteristic size of 25 μm or larger is about 0 to about 600 per mL, for example, about 0 to about 100, about 0 to about 10, about 0 to about 3, about 0 to about 1, about 0 to about 0.5, or about 0 to about 0.1 per mL. Exemplary methods for measuring SvP include analysis of therapeutic biological products using a Coulter Counter, HIAC Royco, or microfluidic imaging system after reconstitution and dilution of the therapeutic biological product to standard concentrations, e.g., about 100 mg / mL or about 1 mg / mL. In some embodiments, when dissolved in an aqueous liquid, the composition has a concentration of insoluble sub-visible particles (SvP) of particles larger than about 10 μm, ranging from about 0 to about 100,000,000 per mL. In certain embodiments, when dissolved in an aqueous liquid, the composition has a concentration of insoluble sub-visible particles (SvP) of particles larger than about 10 μm, ranging from about 0 to about 6000 per mL. In certain embodiments, when dissolved in an aqueous liquid, the composition has a concentration of insoluble sub-visible particles (SvP) of particles larger than about 25 μm, ranging from about 0 to about 600 per mL.In certain embodiments, the composition, when dissolved in an aqueous liquid, is substantially free of insoluble sub-visible particles (SvP).

[0097] In certain embodiments, when dissolved in an aqueous liquid, the composition has a concentration of insoluble sub-visible particles (SvP) having a characteristic size greater than about 10 μm, ranging from about 0 to about 100,000,000 per mL. In some embodiments, when dissolved in an aqueous liquid, the composition has a concentration of insoluble sub-visible particles (SvP) having a characteristic size greater than about 10 μm, ranging from about 0 to about 6,000 per mL. In some embodiments, when dissolved in an aqueous liquid, the composition has a concentration of insoluble sub-visible particles (SvP) having a characteristic size greater than about 25 μm, ranging from about 0 to about 600 per mL. In certain embodiments, the aqueous liquid is water, an aqueous buffer, or a physiologically relevant aqueous liquid.

[0098] In some embodiments, insoluble particulate matter with a characteristic size of approximately 100 nm to 1 μm that persists even after dissolution in an aqueous liquid is referred to as submicron particles (SMPs), often known as nanoparticles. The presence of such SMPs is thought to contribute to immunogenicity and should therefore be avoided to minimize such effects. Quantitatively, SMPs are present in quantities of approximately 0 to 5 × 10⁶ per mL. 12 For example, approximately 0 to approximately 0.5 × 10 per 1 mL 12 Approximately 0 to 50 x 10 per 1 mL 9 Approximately 0 to 10 x 10 per 1 mL 9 Approximately 0 to 5 x 10 per 1 mL 9 Approximately 0 to 0.5 × 10 per 1 mL 9 Approximately 0 to 50 x 10 per 1 mL 6 Approximately 0 to 1 x 10 per 1 mL 6They are present in amounts of approximately 0 to 500,000, 0 to 200,000, 0 to 100,000, 0 to 10,000, 0 to 5,000, or 0 to 1,000 per mL. Exemplary methods for quantitatively measuring SMPs include analysis of therapeutic biological products by NanoSight after reconstitution and dilution of therapeutic biological products to standard concentrations, e.g., about 100 mg / mL, about 1 mg / mL, or about 1 μg / mL; microfluidic imaging systems; asymmetric field flow fractionation (AF4 MALS) coupled to multi-angle laser light scattering; dynamic light scattering (DLS); or FLOWCAM® imaging. Qualitatively, SMPs are within the range corresponding to the starting monomer therapeutic biological product solution. In certain embodiments, the composition, when dissolved in an aqueous liquid, is substantially free of submicron particles (SMPs).

[0099] In certain embodiments of the present disclosure described herein, the particles and compositions have improved stability of the hyaluronan degrader compared to aqueous compositions containing the hyaluronan degrader in a soluble form (see, for example, Example 3). A “stable” composition is one in which all therapeutic biological agents and hyaluronan degraders within it essentially retain their physical and / or chemical stability and / or biological activity, for example, when stored at the intended storage temperature, e.g., 4–40°C, while the container is closed. It is desirable that the composition essentially retains its physical and chemical stability, as well as its biological activity, during storage. The storage period is generally selected based on the intended shelf life of the composition. Furthermore, the composition should be stable after freezing (e.g., down to -70°C) and thawing, for example, after 1, 2, or 3 cycles of freezing and thawing. Various analytical techniques for measuring protein stability are available in the art and have been reviewed, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured at a selected temperature and over a selected period of time. Stability can be evaluated qualitatively and / or quantitatively in a variety of different ways, including evaluation of aggregate formation (e.g., by size exclusion chromatography and / or visual inspection); evaluation of charge heterogeneity using cation exchange chromatography or capillary zone electrophoresis; amino-terminated or carboxyl-terminated sequence analysis; mass spectrometry; SDS-PAGE analysis comparing reduced and intact antibodies; peptide mapping (e.g., trypsin or LYS-C) analysis; and evaluation of the biological activity or antigen-binding function of antibodies. In some embodiments, the therapeutic biological agent and hyaluronan degrader in the composition are stable for at least one month. In some embodiments, the therapeutic biological agent and hyaluronan degrader in the composition are stable for at least three months.In certain embodiments, the therapeutic biological agent in the composition is stable for at least 3 months. In certain embodiments, the therapeutic biological agent and the hyaluronan degrader in the composition are stable at 40°C for at least 3 months.

[0100] Multiple particles comprising at least one protein, for example, the therapeutic biological agents described herein, are used in many ways, as well as in, for example, International Applications PCT / US2017 / 063150 (Publication WO2018 / 098376), PCT / US2018 / 043774 (Publication WO2019 / 023392), PCT / US2019 / 033875 (Publication WO2019 / 226969), PCT / US2020 / 15957 (Publication WO2020 / 160323), PCT / US2020 / 0 The particles disclosed in 50508 (Publication No. WO2021 / 050953), PCT / US2021 / 16878 (WO2021 / 158959), PCT / US2021 / 018806 (WO2021 / 168271), PCT / US2021 / 027755 (WO2021 / 212019), and PCT / US2022 / 072755 (WO2022 / 256840) can be prepared and characterized in any way of forming the particles disclosed therein, the contents of each of these are incorporated herein by reference in their entirety.

[0101] Each of the elements of the Disclosure is described herein as including multiple embodiments, but unless otherwise indicated, each embodiment of a given element of the Disclosure may be used in conjunction with each embodiment of other elements of the Disclosure, and each such use is intended to form a separate embodiment of the Disclosure.

[0102] Other suitable modifications and adaptations to the compositions described herein will be readily apparent from the description of the disclosure contained herein in light of information known to those skilled in the art, and will be understood by those skilled in the art that they can be implemented without departing from the scope of the disclosure or any embodiment thereof.

[0103] Pharmaceutical composition of the present disclosure Provided herein are stable, pharmaceutically effective compositions comprising a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein substantially all of the particles comprise at least one therapeutic biological agent or a salt thereof, and a hyaluronan degrader. The compositions retain their particle size distribution and hyaluronidase activity after long-term storage for at least one month. Stable, pharmaceutically effective compositions can be used to treat diseases or conditions. In particular, the stable, pharmaceutically effective compositions provided herein are formulated for subcutaneous administration.

[0104] Subcutaneously administered preparations offer advantages for home care therapy and are therefore desired to be stable, readily available, and pharmaceutically effective compositions comprising multiple particles suspended in a pharmaceutically acceptable liquid carrier, wherein substantially all of the particles contain at least one therapeutic biological agent or a salt thereof, and the composition also contains a hyaluronan degrader. Therapeutic biological agents used in treatment are generally exposed to a variety of conditions during processing and storage, including temperature fluctuations. For the suspension preparation to be effective, the pharmaceutically effective composition should maintain high protein concentrations and sufficient activity for both the therapeutic biological agent and hyaluronidase in the final readily available suspension preparation. Therefore, pharmaceutically effective compositions of therapeutic biological agents and hyaluronidase should be provided as stable particle suspensions that have the ability to be stored for long periods without degradation. Provided herein are stable, pharmaceutically effective compositions comprising multiple particles suspended in a pharmaceutically acceptable liquid carrier, wherein substantially all of the particles contain at least one therapeutic biological agent or a salt thereof, and a hyaluronan degrader. Pharmacologically effective compositions can be provided in dosage forms that can be used for direct subcutaneous administration. The bioavailability and exposure of subcutaneously administered suspension formulations can be increased in the presence of hyaluronidase (see, for example, Figure 16).

[0105] In certain embodiments, the Disclosure relates to a composition, for example, a pharmaceutically effective composition comprising a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein the particles (e.g., substantially all of the particles) comprise a therapeutic biological agent (e.g., a therapeutic biological agent disclosed herein) and a hyaluronan degrader (e.g., a hyaluronan degrader disclosed herein).

[0106] In some embodiments disclosed herein, the pharmaceutically effective composition comprises a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, substantially all of which comprise at least one therapeutic biological agent or a salt thereof and a hyaluronan degrader, and the concentration of the therapeutic biological agent or salt thereof in the composition is greater than about 250 mg / mL.

[0107] In some embodiments, administration of particles containing hyaluronidase, when administered by subcutaneous syringe injection, allows for increased bioavailability and fluid dispersion of the composition. As disclosed herein, the bioavailability and exposure of a subcutaneously administered suspension formulation can be increased by at least 20% (see, for example, Figure 16). As shown in Example 3, a composition comprising particles having at least one therapeutic biological agent and a hyaluronan degrader showed improved bioavailability and exposure when administered subcutaneously.

[0108] In some embodiments, the concentration of the hyaluronan degrading agent in the composition is less than approximately 0.5 mg / mL, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01 mg / mL. In a particular embodiment, the concentration of the hyaluronan degrading agent in the composition is less than approximately 0.5 mg / mL.

[0109] As used herein, the term “pharmaceutically acceptable liquid carrier” means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid, diluent, excipient, or solvent. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the composition and is not harmful to the patient. Those skilled in the art can dilute or increase the volume of therapeutic biological preparations using inert substances in particles. These diluents may include carbohydrates, particularly trehalose, mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextran, and starch. Certain inorganic salts, including calcium triphosphate, magnesium carbonate, and sodium chloride, may also be used as fillers. Some commercially available diluents include Fast-Flo, Emdex, STA-Rx 1500, Emcompress, and Avicell.

[0110] In some embodiments, the pharmaceutical composition for administration includes a non-aqueous solution of the active therapeutic biological preparation in a water-soluble form. Preferably, a suspension of the active therapeutic biological preparation can be prepared as a suitable oily injection composition. Suitable lipophilic solvents or vehicles include fatty oils (e.g., sesame oil, corn oil), or fatty acid esters (e.g., ethyl oleate or triglycerides), or liposomes. Appropriate viscosity can be maintained by maintaining the required particle size in the case of injection, and by the use of flocculants. Prevention of microbial activity can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal.

[0111] This disclosure includes the use of “pharmaceutically acceptable salts” or “salts” of therapeutic biological products in compositions of this disclosure. As used herein, the terms “pharmaceutically acceptable salts” or “salts” include, for example, salts derived from inorganic or organic acids, including hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, phosphoric acid, formic acid, acetic acid, lactic acid, maleic acid, fumaric acid, succinic acid, tartaric acid, glycolic acid, salicylic acid, citric acid, methanesulfonic acid, benzenesulfonic acid, benzoic acid, malonic acid, trifluoroacetic acid, trichloroacetic acid, naphthalene-2-sulfonic acid, or other acids. In some embodiments, the form of a pharmaceutically acceptable salt may include forms in which the ratio of molecules containing the salt is not 1:1. For example, a salt may contain more than one inorganic or organic acid molecule per molecule of base, such as two hydrochloric acid molecules per molecule of therapeutic biological product. As another example, a salt may contain less than one inorganic or organic acid molecule per base molecule, such as a therapeutic biological preparation with two molecules per base molecule of tartaric acid. In certain embodiments, the salts contemplated in this disclosure include, but are not limited to, arginine, venentamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydravamin, 1H-imidazole, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, or zinc salts. In some embodiments, the salts contemplated in this disclosure include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts. In further embodiments, the salts intended by this disclosure include, but are not limited to, alkylammonium salts, dialkylammonium salts, trialkylammonium salts, or tetraalkylammonium salts. The pharmaceutically acceptable salts may also exist as various solvates with, for example, water or ethanol. Mixtures of such solvates can also be prepared. In some embodiments, the proteinaceous composition may be formulated in neutral or salt form.Pharmaceutically acceptable salts include acid addition salts (formed using free amino groups of proteins), which are formed using inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, and mandelic acid. Salts formed using free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, and procaine.

[0112] In some embodiments, particles can be suspended in a non-aqueous liquid carrier to form a non-aqueous, pharmaceutically acceptable composition. Importantly, the process for producing a non-aqueous composition having at least one therapeutic biological formulation does not significantly alter the structure or bioactivity of the biological formulation described herein. In some embodiments, the liquid carrier is non-aqueous. In certain embodiments, the non-aqueous liquid carrier is an organic solvent. In some embodiments, the non-aqueous liquid carrier comprises at least two organic solvents.

[0113] In some embodiments, the organic solvent is benzyl benzoate, coconut oil, cottonseed oil, fish oil, grape seed oil, hazelnut oil, hydrogenated vegetable oil, olive oil, coconut seed oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, sunflower oil, walnut oil, corn oil, acetone, ethyl acetate, ethyl lactate, dimethylacetamide, dimethyl isosorbide, dimethyl sulfoxide, glycoflore, diglyceride, methyl tert-butyl ether, N-methylpyrrolidone, perfluorodecalin, polyethylene glycol, 2-pyrrolidone, tetrahydrofurfuryl alcohol, diglycerides, triglycerides, medium-chain triglycerides (MCTs), caproic acid, caprylic acid, capric acid, lauric acid, ethyl laureate, triglycerides of fractionated vegetable fatty acids C8 and C10, propylene glycol of saturated vegetable fatty acids C8 and C10 Licor diester (PGD), ethyl oleate, ethyl caprate, dibutyl adipate, fatty acid esters, hexanoic acid, octanoic acid, triacetin, diethyl glycol monoether, gamma-butyrolactone, eugenol, clove bud oil, citral, limonene, polyoxyl 40 hydrogenated castor oil, polyoxyl 35 castor oil, simple alcohols, e.g., ethanol, octanol, hexanol, decanol, propanol, butanol, gamma-butyrolactone, tocopherol, octa-fluoropropane, (perfluorohexyl)octane, n-acetyltryptophan, ethyl laurate, methyl caprylate, ethyl caprylate, methyl caprate, methyl myristate, methyl oleate, methyl linoleate, dimethyl adipate, dibutyl suberate, diethyl sebacate, macadamia nut fatty acid ethyl (ethyl)This includes macadamiate, trimethylolpropane triisostearate, isopropyl laurate, isopropyl myristate, diethyl succinate, polysorbate esters, ethanolamine, propanoic acid, citral, anisole, anethole, benzaldehyde, linalool, caprolactone, phenol, thioglycerol, dimethylacetamide, diethylene glycol monoethyl ether, solketal, isosorbide dimethyl ether, ethyl formate, hexyl ethyl acetate, propylene glycol dicaprylate, caprylic acid triglyceride, ethyl linoleate, ethyl linolenate, or combinations thereof. Medium-chain triglycerides (MCTs) contain fatty acids with a chain length of 6 to 12 carbon atoms. These include caproic acid (C6), caprylic acid (C8), capric acid (C10), and lauric acid (C12). In some embodiments, the organic solvent is ethyl oleate, diglycerides, triglycerides, migliol, ethyl macadamia nut fatty acid, ethyl caprate, diethyl succinate, diethylene glycol monoethyl ether, propylene glycol dicaprylate, caprylic triglyceride, ethyl linoleate, ethyl linolenate, medium-chain triglycerides (MCT), sesame oil, propylene glycol diesters (PGD) of saturated vegetable fatty acids C8 and C10, triacetin, or a combination thereof. In certain embodiments, the organic solvent is ethyl oleate and sesame oil. In some embodiments, the organic solvent is propylene glycol diesters (PGD) of saturated vegetable fatty acids C8 and C10. In some embodiments, the organic solvent is ethyl oleate.

[0114] In some embodiments, the concentration of the therapeutic biological agent in the composition disclosed herein is greater than about 250 mg / mL, for example, greater than about 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, or 800 mg / mL. In certain embodiments, the concentration of the therapeutic biological agent in the composition is greater than about 800 mg / mL. In some embodiments, the concentration of the therapeutic biological agent in the composition is greater than about 400 mg / mL. In certain embodiments, the concentration of the therapeutic biological agent in the composition is greater than about 500 mg / mL. In some embodiments, the concentration of the therapeutic biological agent in the composition is greater than about 600 mg / mL. In certain embodiments, the concentration of the therapeutic biological agent in the composition is greater than about 700 mg / mL.

[0115] Injectable particle suspensions can exhibit sedimentation variability, which can affect the use or administration of the drug composition. In particular, injectable particle suspensions of high concentrations, low volumes, and low syringe extrusion forces of therapeutic biological products may settle or precipitate from the suspension medium over time, and therefore require pre-mixing or resuspension before injection. Sedimentation of high particle concentrations at low delivery volumes can also lead to high injection forces and propagate the degradation of the therapeutic biological product in the composition. As used herein, the terms “settling” or “deposition” refer to the process by which particles settle or densely accumulate at the bottom of a container closure, such as a vial, cartridge, syringe, or portable drug delivery injection device. Sedimentation of densely accumulated particles generally leads to excessively high injection forces or syringe occlusion and requires manual stirring or pre-mixing before administration of the composition.

[0116] Aggregation, or "particle agglutination," is a phenomenon arising from the interrelationship between interfacial chemistry governing particle-particle interactions and environmental conditions. As used herein, agglutination volume is a measure of the amount of particle agglutination occupied by particle dispersion, expressed as a percentage of the total fluid volume. More specifically, agglutination volume (F) is the ratio of the volume of particle agglutination (Vu) to the total fluid volume (Vo). Agglutination volume can decrease over time based on the sedimentation properties of particle agglutination. In an agglutinated suspension, particles undergo collisions that lead to particle agglutination or agglutination, which increases in size, and thus increases, for example, the agglutination volume. The term "agglutination" refers to a cluster of particles that is loosely agglutinative. The loose network of particle agglutination maintains sufficient interparticle distance to prevent particle sedimentation, which can lead to high injection forces. When the agglutination volume is equal to 1, the yield stress (the stress at which the agglutination volume deforms) can be measured to indicate the shear stress required to allow the suspension to flow. Measurements for yield stress can be obtained using a parallel plate rheometer (ANTON PAAR® MCR 92). An increase in yield stress indicates an increase in agglutination. Other approaches for measuring agglutination include, but are not limited to, laser diffraction, relaxation NMR, microscopy, and small-angle scattering.

[0117] In the compositions disclosed herein, the agglomeration process can be controlled by the use of a flocculant. A flocculant, or coagulant, is a chemical substance that promotes aggregation by causing suspended particles in a liquid to agglomerate, thereby forming a clump. In non-aqueous particle suspensions, the breakdown and redispersion of the clump are governed by the shear stress imparted to the agglomerated particle system. Shear stress is caused by a force acting on the surface of a material, leading to deformation. In these systems, shear can result from the movement of particles or clumps through a velocity gradient in a non-aqueous suspension medium or near any interface (e.g., container closure, other particles, etc.). Shear stress can be introduced into the agglomerated volume mechanically, for example, and can be controlled experimentally.

[0118] The compositions disclosed herein, comprising multiple particles suspended in a pharmaceutically acceptable liquid carrier, further comprising a flocculant, are generally high-concentration, low-volume, and low-syringe-extraction-force injectable particle suspensions of therapeutic biological agents and hyaluronane degraders, enabling administration without requiring manual stirring or pre-mixing before injection or administration of the composition. The compositions beneficially maintain a stable aggregate volume to avoid particle sedimentation, which can lead to clogging of syringes and portable drug delivery injection devices. In some embodiments, the compositions further comprise a flocculant.

[0119] In the compositions described herein, the coagulation process can be controlled by the use of a flocculant that allows the administration of the particle composition without requiring manual stirring or pre-mixing before injection or administration. The flocculant may be nonionic or ionic. Generally, ionic flocculants interact with charged particles; for example, anionic flocculants interact with positively charged particles, and cationic flocculants interact with negatively charged particles. In some embodiments, the flocculant is ionic. In some embodiments, the ionic flocculant is magnesium stearate, sodium dodecyl sulfate, sodium stearate, cetyltrimethylammonium bromide, lecithin, or a combination thereof. In certain embodiments, the flocculant is nonionic. As disclosed herein, nonionic flocculants interact with charged particles. In certain embodiments, the nonionic flocculant is polysorbate (polysorbate 80, polysorbate 60, polysorbate 20, e.g., Tween 80, Tween 60, Tween 20), alkylphenol ethoxylate, glycerol, polyoxyethylated castor oil, doxate, decyl glucoside, nonoxynol-9, sorbitan ester, sorbitan monooleate, ethanolamine, polyoxyl 35 castor oil, poloxyl 40 hydrogenated castor oil, carbomer 1342, corn oil mono-di-triglyceride, polyoxyethylated oleiceride, poloxamer, or a combination thereof. In some embodiments, the nonionic flocculant is polysorbate, alkylphenol ethoxylate, sorbitan ester, poloxamer, or a combination thereof. In certain embodiments, the nonionic flocculant is polysorbate or sorbitan ester. In some embodiments, the addition of a flocculant to a particle suspension does not substantially increase the viscosity of the composition. As disclosed herein, the addition of a flocculant to a particle suspension increases the aggregated volume of the particle suspension. In certain embodiments, the addition of a flocculant to a particle suspension increases the stability of the aggregated volume for at least one month. In some embodiments, the addition of a flocculant to a particle suspension reduces the syringe extrusion force.In some embodiments, the addition of a flocculant to a particle suspension improves the pharmacokinetics (PK) of administration (e.g., subcutaneous administration) of a suspension of fine particles (e.g., Ab fine particles) and exhibits higher bioavailability than administration (e.g., subcutaneous administration) of a composition containing multiple particles, including at least one therapeutic biological agent. In some embodiments, the addition of a flocculant to a particle suspension increases the stability of proteins in the particles.

[0120] In some embodiments, the compositions described herein use a flocculant concentration in the composition of less than about 50 mg / mL, for example, less than about 45, 40, 35, 30, 25, 20, 15, 10, 5, 3, 1, 0.5, 0.1, 0.05, or 0.01 mg / mL. In some embodiments, the flocculant concentration in the composition is less than about 10 mg / mL. In some embodiments, the flocculant concentration in the composition is less than about 5 mg / mL. In some embodiments, the flocculant concentration in the composition is less than about 3 mg / mL. In certain embodiments, the flocculant concentration in the composition is less than about 1 mg / mL. In some embodiments, the flocculant concentration in the composition is less than about 0.1 mg / mL. In certain embodiments, the flocculant concentration in the composition is less than about 0.01 mg / mL.

[0121] Viscosity can play a significant role in the handling and administration of injectable products. For suspension products, high-viscosity drug products can be difficult to deliver via needles (e.g., 27-gauge needles) because they require greater force to operate the injection device, such as a syringe or portable drug delivery device. Alternatively, using larger gauge needles or requiring longer injection times may reduce patient compliance with the therapy.

[0122] The term "viscosity" is used to describe the properties of a fluid that act to resist shear flow. For the purposes of this disclosure, viscosity can be determined using a rheometer equipped with a cone and plate (2° / 40 mm), e.g., an AR-G2 rheometer (TA Instruments, USA), at a specified shear rate at 25°C. In certain embodiments, viscosity is measured at the shear rate in the Newtonian region. In some embodiments, viscosity is measured at 100 s -1 Or a shear rate exceeding that, for example, 1000 s -1 , or 1000s -1 Over, or 10,000s -1 Measured in ultra-low viscosity. As used herein, the term "low viscosity" describes compositions having a viscosity of less than approximately 100 mPa·s, such as liquid carriers.

[0123] In some embodiments, the composition is less than approximately 200 mPa·s, less than approximately 150 mPa·s, less than approximately 125 mPa·s, less than approximately 100 mPa·s, less than approximately 95 mPa·s, less than approximately 90 mPa·s, less than approximately 85 mPa·s, less than approximately 80 mPa·s, less than approximately 75 mPa·s, less than approximately 70 mPa·s, less than approximately 65 mPa·s, less than approximately 60 mPa·s, less than approximately 55 mPa·s, less than approximately 50 mPa·s, less than approximately 45 mPa·s, less than approximately 40 mPa·s, less than approximately 35 mPa·s, less than approximately 30 mPa·s, less than approximately 25 mPa·s, less than approximately 20 mPa·s The viscosity is less than approximately 19 mPa·s, less than approximately 18 mPa·s, less than approximately 17 mPa·s, less than approximately 16 mPa·s, less than approximately 15 mPa·s, less than approximately 14 mPa·s, less than approximately 13 mPa·s, less than approximately 12 mPa·s, less than approximately 11 mPa·s, less than approximately 10 mPa·s, less than approximately 9.5 mPa·s, less than approximately 9 mPa·s, less than approximately 8.5 mPa·s, less than approximately 8 mPa·s, less than approximately 7.5 mPa·s, less than approximately 7 mPa·s, less than approximately 6.5 mPa·s, less than approximately 6 mPa·s, less than approximately 5.5 mPa·s, or less than approximately 5 mPa·s (1 millipascal second). Furthermore, viscosity can be controlled using a mixture of liquids. The units "mPa·s" and "cP" are used interchangeably in the broadest sense herein.

[0124] In some embodiments, the composition has a viscosity of less than about 100 mPa·s. In some embodiments, the composition has a viscosity of less than about 80 mPa·s. In a particular embodiment, the composition has a viscosity of less than about 50 mPa·s. In some embodiments, the composition has a viscosity of less than about 40 mPa·s. In a particular embodiment, the composition has a viscosity of less than about 30 mPa·s. In a particular embodiment, the composition has a viscosity of less than about 25 mPa·s. In some embodiments, the composition has a viscosity of less than about 20 mPa·s. In a particular embodiment, the composition has a viscosity of less than about 15 mPa·s. In some embodiments, the composition has a viscosity of less than about 10 mPa·s. In a particular embodiment, the composition has a viscosity of less than about 5 mPa·s.

[0125] As described herein, a higher coagulation volume indicates increased agglutination or less particle sedimentation, and therefore improved injection force performance due to a more uniform dispersion of particles in the liquid carrier. In some embodiments, the composition has a coagulation volume of more than about 50% after initial mixing, for example, more than 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more than about 98% after initial mixing. In some embodiments, the composition has a coagulation volume of more than about 70% after initial mixing. In certain embodiments, the composition has a coagulation volume of more than about 80% after initial mixing. In certain embodiments, the composition has a coagulation volume of more than about 85% after initial mixing. In certain embodiments, the composition has a coagulation volume of about 100% after initial mixing. Initial mixing refers to the homogenization of the initial heterogeneous particle suspension with the flocculant by the movement of the bulk that creates the coagulation volume.

[0126] In some embodiments, the aggregated volume is reduced by less than about 10%, e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% after at least one month under closed storage conditions at below about 40°C. In some embodiments, the aggregated volume is reduced by less than about 7% after at least one month under closed storage conditions at below about 40°C. In certain embodiments, the aggregated volume is reduced by less than about 5% after at least one month under closed storage conditions at below about 40°C. In some embodiments, the aggregated volume is reduced by less than about 3% after at least one month under closed storage conditions at below about 40°C. In certain embodiments, the aggregated volume is reduced by less than about 1% after at least one month under closed storage conditions at below about 40°C. In some embodiments, the aggregated volume remains substantially suspended in the liquid carrier for at least one month. In some embodiments, the composition has substantially the same aggregated volume for at least one month.

[0127] A dense (1.32 g / cm³) material with a controllable size distribution (polydispersion index < 0.2). 3The generation of round particles can be achieved using various therapeutic biological formulations described herein. Focused ion beam scanning electron microscopy (FIB-SEM) can be used to determine whether the particles contain void spaces, which is crucial for achieving a high protein content in the composition, and X-ray photoelectron spectroscopy (XPS) can be used to control the radial distribution of the particles. In certain embodiments, the particle size may be about 10 to about 80 μm, which is preferable for achieving a low viscosity composition but small enough to prevent clogging in a 27-gauge needle syringe. For example, by filling a pre-filled syringe with a flocculant and particles at a protein concentration of about 400 mg / mL containing a hyaluronan degrading agent at about 0.5 mg / mL in an ethyl oleate (EO) liquid carrier, a therapeutic composition can be formed with a viscosity of about 20 mPa·s (corresponding to an injection force of about 4 N), which can be stored for at least one month without substantially changing the aggregate volume, viscosity, or injection force (e.g., the particles remain substantially suspended in the liquid carrier for at least one month). In some embodiments, the composition has substantially the same aggregate volume for at least one month. In some embodiments, multiple particles and the flocculant remain substantially suspended in the liquid carrier for at least three months. In certain embodiments, the injection force remains substantially the same for at least three months under closed storage conditions at about 25°C. In some embodiments, the yield stress remains substantially the same for at least one month under closed storage conditions at about 25°C. In certain embodiments, the addition of a flocculant to the composition prevents particle sedimentation and needle clogging.

[0128] As described herein, the characterization of the structural stability of particles in the compositions was achieved using size exclusion chromatography (SEC), differential scanning fluorescence spectroscopy (DSF), circular dichroism (CD), cation exchange chromatography (CIEX), and sub-visible particle (SvP) analysis. In addition, pharmacokinetic (PK) and immunogenicity studies were used to analyze the conservation of biological activity.

[0129] Furthermore, as described herein, SEC data confirmed that minimal aggregate formation was observed during processing for at least 3 months compared to a labeled formulation containing aggregates (aqueous mAb as an FDA-approved formulation) compared to a recombined particle composition containing 4.7% high molecular weight aggregates, 94.6% monomer data, and 0.72% low molecular weight data as determined by SEC (protein content 507 mg / mL). See Example 3. After 30 days of storage at 40°C, DSF showed a thermal shift of less than 1°C across the entire sample, and CD could not detect any difference in secondary structure (beta-sheet percentage). CIEX was used to analyze the protein charge variants as required by regulation (ICH Q6B) to ensure that no chemical modifications occurred during preparation and storage as described herein. In certain embodiments, the composition may be less susceptible to chemical modifications during storage than the FDA-labeled formulation. This is because the protein is more stable in the solid state as particles.

[0130] After 30 days of storage at 40°C, preservation of biological activity was demonstrated by flow cytometry assays. No discernible differences were apparent between the FDA-labeled formulations and their compositions described herein. As shown in Examples 1 and 2 herein, the biological activity of the FDA-labeled formulations decreased significantly after storage, but no decrease in activity was observed for the particles in the compositions.

[0131] Prior to the in vivo study, the particles used in the composition were obtained under substantially sterile conditions. Bacterial endotoxin levels were approximately three orders of magnitude lower than the accepted injection standard (0.05 EU / mg to 0.25 EU / mg). In addition, microbial growth assays did not show any observable growth.

[0132] The compositions described herein are comparable to aqueous FDA-labeled formulations in terms of rat pharmacokinetics (PK) and SC clearance (mouse). The PK profiles are shown in Example 3, where the mAb and hyaluronidase microparticle suspension (SC injection) exhibits higher bioavailability than the mAb microparticle suspension. See Figure 16. The in vivo solubility behavior of the particles has been shown to ensure that the compositions can eliminate injection sites at an increased rate compared to standard aqueous formulations, as undissolved particles may potentially trigger an immunogenic response. In some embodiments, the compositions have improved pharmacokinetics (PK) compared to compositions containing multiple particles, each containing at least one therapeutic biological agent. See Figure 16. The area under the curve (AUC) from zero to infinity represents the total drug exposure over time. Peak concentration is a pharmacokinetic measure used to determine drug dosage. Maximum concentration (Cmax) is the highest concentration of the drug in the blood, cerebrospinal fluid, or target organ after the dose has been administered. Tmax is the time it takes for a drug (e.g., an oral drug) to reach its maximum concentration (Cmax) after administration. Tmax is governed by the rate of drug absorption and the rate of drug elimination. In certain embodiments, the composition has at least 20% improved AUC, Cmax, and / or Tmax compared to a composition comprising multiple particles, each containing at least one therapeutic biological agent.

[0133] This disclosure relating to a highly concentrated composition comprising a flocculant and a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein substantially all of the particles (e.g., substantially all of the particles) contain at least one therapeutic biological agent and a hyaluronan degrader, and the composition, when dissolved in water, a buffer, or other physiologically relevant aqueous liquid, e.g., a biological fluid in a patient's body, has substantially similar immunogenicity to a similar aqueous composition containing a monomer therapeutic biological agent. Where used herein, the term “physiologically relevant” can be applied as something that may be encountered within a mammal or human. Those skilled in the art will be able to determine the set of conditions most appropriate for testing, depending on the final use of the composition described herein. In some embodiments, the composition, when dissolved in an aqueous liquid, has substantially similar immunogenicity to an aqueous composition containing at least one therapeutic biological agent in a soluble form, e.g., a monomer form. In certain embodiments, the composition is substantially non-immunogenic. In certain embodiments, the aqueous liquid is water, an aqueous buffer, or a physiologically relevant aqueous liquid.

[0134] As disclosed herein, the term “immunogenicity” refers to the induction of an immune response by an injected composition of a therapeutic biological agent (antigen), while “antigenicity” refers to the reaction of a composition of a therapeutic biological agent with existing antibodies. Collectively, antigenicity and immunogenicity are referred to as “immunoreactivity.” In certain embodiments, a composition has substantially similar immunogenicity to an aqueous composition containing at least one therapeutic biological agent in a soluble form, e.g., monomeric form. In certain embodiments, a composition is substantially non-immunogenic when subjected to the presence of a non-immune response, for example, based on repeated-dose anti-drug antibody immunogenicity analysis.

[0135] The ratio between toxicity and therapeutic effect of a particular composition, for example, human IgG, is its therapeutic index, and LD50. 50 (Compound lethal dose in 50% of the population) and ED 50It can be expressed as a ratio between (the amount of the compound effective in 50% of the population). Compositions exhibiting a high therapeutic index are preferred. Therapeutic index data obtained from cell culture assays and / or animal studies, e.g., from human IgG, can be used in the formulation of various dosages for use in humans and are known in the art. Doses of such compounds are preferably ED with little or no toxicity. 50 The plasma concentration range includes [the substance]. The dosage may vary within this range depending on the dosage form used and the route of administration utilized, e.g., subcutaneous injection. See, for example, Fingl et al., In: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Ch.1, p.1, 1975. The exact composition, route of administration, and dosage may be selected by the individual physician, taking into account the patient's condition and the specific method by which the composition is delivered.

[0136] In some embodiments, the composition has substantially similar toxicity to an aqueous composition containing at least one therapeutic biological agent in a soluble form, e.g., a monomeric form. In some embodiments, the composition has reduced toxicity to an aqueous composition containing at least one therapeutic biological agent in a soluble form, e.g., a monomeric form. In certain embodiments, the composition is substantially nontoxic, for example, based on topical tolerability and clinical observational analysis.

[0137] Method of Disclosure Formulations of therapeutic biological agents and hyaluronane degraders are generally in liquid form. However, storing co-formulations in liquid form presents challenges in terms of chemical and physical stability during storage, which limits the shelf life of ready-to-use preparations. Formulating therapeutic biological agents and hyaluronane degraders in particle suspensions takes advantage of the storage stability in solid form, the ease of administration of high-concentration, low-volume injectable particle suspensions, and the ability to supply particle suspensions in convenient pre-filled, ready-to-use preparations without reconstitution. Due to the increased stability during storage, the particle suspensions of therapeutic biological agents and hyaluronane degraders described herein can contain high protein concentrations exceeding approximately 250 mg / mL without causing any adverse effects on biological activity due to coagulation, fragmentation, and / or changes in charge variants of the therapeutic biological agent during long-term storage.

[0138] An example of a general protocol for preparing protein particles using hyaluronidase is as follows:

[0139] Aqueous protein solutions with concentrations of 50–180 mg / mL can be prepared. An aqueous solution of excipients and hyaluronidase (e.g., a stock concentration of hyaluronidase of 2000 U / mL, 0.000051–8.81% w / w; particle load 375–750 mg / mL) can be added to the aqueous protein solution to form an aqueous feed solution. The aqueous feed solution can be filtered to remove any exogenous solids from the resulting aqueous "feed" solution. In some embodiments, the hyaluronidase may be in an aqueous carrier. For example, a polymer may be added to a carrier liquid such as water together with the hyaluronidase. Aqueous droplets can be produced by mixing the protein feed and a dehydrating solvent (e.g., n-butyl acetate or pentanol) at different flow rates (1 mL / min–10 L / min). The aqueous droplets can be dehydrated for a dehydration time determined by the volume of the dehydrating solvent, the rpm of the mixture, and the flow rate to form a protein particle suspension. Protein particles can be separated from the liquid mixture using methods such as filtration, centrifugation, and decanting. Aqueous liquids and dehydrated solvents can be removed as residual solvents. A flocculant can be added to form a non-precipitating suspension. Examples of flocculants include lecithin and / or PS80.

[0140] Residual solvent in protein particles can be removed using, for example, vacuum drying, gas drying, gas sparging, or extraction solvents, and the water content in the particles can be adjusted using, for example, humid gas or extraction solvents. Some of the protein particles can be dissolved in DI water and incubated for complete redissolution for characterization. The solution can be analyzed to measure protein quality using, for example, SvP analysis with FLOWCAM®, as well as soluble coagulation (e.g., SEC), fragmentation (e.g., SEC), changes in charge variants (e.g., ion exchange chromatography (e.g., strong cation exchange chromatography (SCEX)) analysis using HPLC-SEC), and protein concentration. Protein concentrations in the range of 200 mg / mL to 800 mg / mL may contain at least 60–93% (w / w) particle protein content. Particles with a maximum protein content of 85% showed improved stability compared to the initial aqueous supply solution. Generally, the amount of hyaluronidase in the suspension may be about 20 U / mg of protein. In some embodiments, the amount of hyaluronidase in the suspension may be 0 U / mg to 1000 U / mg per 1 mg of protein.

[0141] Embodiments of non-settling suspensions can prevent the sedimentation of particles that form a reduced cake height in the suspension. Non-settling suspensions can maintain cake height over long-term storage, e.g., days, weeks, months, or years. In some embodiments, non-settling suspensions containing a flocculant such as PS80 can maintain a monomer level of 91.3% over 6 months of accelerated storage at 40°C.

[0142] Preparation of Ab particles in the target size and dispersibility range: Rituximab, trastuzumab, human IgG particles, or bovine IgG particles can be formed under the general protocol described above. The average particle size, D10, D50, and D90 of the particles can be determined to have an average particle size of 5–50 μm and D90 < 60 μm. Particles formed using the general protocol with trastuzumab and hyaluronidase may be smooth and spherical and lack any void spaces. In some embodiments, the roundness of the particles may be 0.99–1.00 or 0.90–1.00.

[0143] Particles containing trastuzumab, rituximab, mixed antibodies, and BSA can be suspended in PGD or EO. Non-precipitating suspensions are particle suspensions having a flocculant such as lecithin or PS80, but are not limited to these. Particle size and span can be formed within a predetermined range so that the suspension can flow through a syringe without clogging. Additionally, the sub-visible particulate matter in the particles has been found to be lower than in any other finished technique, including standard lyophilization.

[0144] Hyaluronidase in particles formed using general procedures can maintain stability over long periods at storage temperatures known in the art, such as 4°C. The stability of the hyaluronidase enzyme in particles was shown to be maintained at approximately 97% under accelerated storage at 40°C for 6 months. The stability of aqueous formulations was shown to be maintained at approximately 91% under the same conditions.

[0145] An example of a general protocol for protein particles containing a hyaluronidase composition: Rituximab, trastuzumab, or IgG (human or bovine) particles containing excipients (e.g., histidine, arginine HCl, PS80, PS20, trehalose, NaCl, sucrose, methionine, proline, sodium phosphate) and hyaluronidase can be combined to form a suspension at a protein concentration of 200–700 mg / mL with a hyaluronidase concentration of up to 1800 or 2000 units / mL (<0.01%) in an organic carrier liquid or a mixture of at least two organic carrier liquids, including but not limited to ethyl oleate, sesame oil, medium-chain triglycerides (MCT), propylene glycol diesters (PGD) of saturated vegetable fatty acids C8 and C10, triacetin, and capryl triglycerides. The protein particles containing hyaluronidase can be added to the organic carrier liquid. A mixer (e.g., a rotor stator) can produce a diluted suspension. The diluted suspension can be sieved through a filter to adjust it to the desired protein concentration. A flocculant can be added to reach a total flocculant concentration of up to 10 mg / mL at a protein concentration of 200–800 mg / mL. Generally, the amount of hyaluronidase in the suspension may be about 20 U / mg per 1 mg of protein. The suspension can be mixed by vortexing and / or mechanical mixing for 1 minute to produce the desired flocculation volume which remains stable for at least 1 month. In some embodiments, the suspension remains stable for at least 6 months in accelerated storage at 40°C. In some embodiments, the suspension remains stable for at least 6 months in storage at 4°C.

[0146] HIgG particle composition: HIgG particles containing hyaluronidase (e.g., 77% protein: 2% histidine: 20% arginine: 1% PS80 (or PS20): 0.006% hyaluronidase) can form a suspension in ethyl oleate. The suspension can be formed by adding the particles to ethyl oleate and producing a diluted suspension using a mixer such as a rotor-stator mixer. The diluted suspension can be sieved through a filter and adjusted to a predetermined concentration of HIgG by centrifugation. Optionally, an aliquot of this suspension was pipetteed with a flocculant (such as PS80) to reach the final total concentration. This suspension can be vortexed and then mechanically mixed for 1 minute to produce a stable suspension (aggregate volume) that maintains consistency for at least 1 month compared to a particle suspension without an agglutinant. In some embodiments, the suspension remains stable for at least 6 months under accelerated storage at 40°C. In some embodiments, the suspension remains stable for at least 6 months under storage at 4°C. HIgG and BIgG can be used interchangeably. Characterization of proteins from the suspension showed improved stability compared to the initial aqueous supply solution.

[0147] The stability of protein and hyaluronidase particle suspensions containing flocculants was evaluated over periods of up to 6 months at 5, 25, and 40°C to determine the stability of the particle suspension compositions compared to protein liquid drug substances (LDS), for example, compared to the initial aqueous supply solution.

[0148] The particles may be suspended in PGD, ethyl oleate, MCT, or a mixture such as ethyl oleate and MCT. In some embodiments, the mixture may be in a 1:1 ratio.

[0149] In some embodiments, LDS samples stored at 40°C for at least 6 months exhibit higher protein coagulation compared to suspensions containing protein particles and flocculant samples stored at the same temperature (40°C).

[0150] The low rate of coagulation in particle suspensions containing flocculants showed improved stability compared to protein LDS. For particle suspensions containing flocculants, protein concentration, hyaluronidase activity, viscosity, and injection force may not change significantly over all storage times and temperatures. The bioavailability of particles containing hyaluronidase may be higher than that of particles without hyaluronidase.

[0151] In some embodiments, after storage at 4°C and 40°C for 3 months, the BSA protein quality attributes (i.e., HMW%, charge variants (basic% and acidic%), and sub-visible particles (>2um)) are similar to those of the aqueous formulation (supply). In some embodiments, formulations with and without hyaluronidase may have similar protein quality, demonstrating that the addition of the enzyme does not affect the quality of the therapeutic protein. Samples with and without hyaluronidase may have similar stability profiles for BSA formulated as a suspension in PGD.

[0152] In some embodiments, when the aqueous formulation (supply) was compared with the suspension formulation (suspension-PGD) and non-precipitating suspensions with flocculant formulations (non-precipitating suspension-PGD, non-precipitating suspension-EO), less than 10% coagulation and substantially no fragmentation were observed after treatment of particles containing both trastuzumab with and without hyaluronidase. Similarly, no substantial changes were observed after treatment. In some embodiments, the suspension exhibited better stability than the aqueous formulation during storage for 6 months.

[0153] In some embodiments, cake height measurements of trastuzumab suspensions compared to non-sedimenting suspensions indicate that adding a flocculant, such as a surfactant like PS80, to the carrier liquid prevents the particles from settling over a 3-month storage period at room temperature. In some embodiments, suspensions without hyaluronidase showed a cake height of 85.6% after 3 months of storage at 25°C, while suspensions containing hyaluronidase showed a cake height of 86.6%. In some embodiments, the cake height of the suspension with a flocculant was 100%.

[0154] In some embodiments, a suspension containing protein particles (e.g., trastuzumab, trastuzumab and rituximab, trastuzumab and hyaluronidase, etc.) and a flocculant has a cake height of about 50% to about 100% (e.g., about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 95% to about 100%, about 80% to about 90%, about 85% to about 90%, etc.) after storage at 25°C for 3 months. In some embodiments, the protein particles contain one or more proteins (e.g., trastuzumab and rituximab) as well as hyaluronidase. In some embodiments, the suspension contains protein particles, a flocculant, and hyaluronidase.

[0155] Cake height measurements of trastuzumab and rituximab (mixed antibody) suspensions compared to non-sedimenting suspensions show that adding PS80 surfactant as a flocculant to the carrier liquid prevents particle sedimentation to 88.2% of the cake height after storage at 25°C for 48 hours.

[0156] Co-formulated particle compositions containing at least two proteins and hyaluronidase can be formed. In some embodiments, particles can be formed using the general procedure described above, having rituximab, trastuzumab, and hyaluronidase. Examples of co-formulated particles include rituximab:trastuzumab and hyaluronidase. The suspension may contain a flocculant such as PS80. In some embodiments, when a flocculant is added to the suspension, no change in aggregate volume may be observed at 4°C or 25°C after at least one month. Furthermore, the injection force may remain substantially the same at approximately 2N (up to 14 seconds) using a 20-gauge 13 mm (half-inch) needle (Japan Bio Products) attached to a 1 mL syringe.

[0157] In some embodiments, particles in suspension may be stable for extended periods. For example, no significant change in protein quality may be observed after storage at 4°C for 3 months. After storage at 40°C for 3 months, the proportion of neutral antibody species may be higher compared to the aqueous formulation (supply), while the amount of SVP may be lower compared to the aqueous formulation (supply). Formulations with and without hyaluronidase may have similar protein quality, indicating that the addition of the enzyme does not affect the quality of the therapeutic protein.

[0158] In some embodiments, the composition (e.g., suspension) comprises particles containing at least about 50 mg / ml (e.g., at least about 100 mg / ml, 150 mg / ml, 200 mg / ml, 250 mg / ml, 300 mg / ml, 350 mg / ml, 400 mg / ml, 450 mg / ml, 500 mg / ml, 550 mg / ml, etc.) of a therapeutic biological agent (e.g., protein, hormone, antibody such as hIgG). In some embodiments, the composition (e.g., suspension) comprises particles containing at least about 50 mg / ml (e.g., at least about 100 mg / ml, 150 mg / ml, 200 mg / ml, 250 mg / ml, 300 mg / ml, 350 mg / ml, 400 mg / ml, 450 mg / ml, 500 mg / ml, 550 mg / ml, etc.) of a therapeutic biological agent (e.g., protein, hormone, antibody such as hIgG) and one or more flocculants.

[0159] In some embodiments, the composition (e.g., suspension) contains particles comprising a therapeutic biological agent (e.g., protein, hormone, antibody such as hIgG) in an amount of about 50 mg / ml to about 1000 mg / ml (e.g., about 100 mg / ml to about 1000 mg / ml, about 200 mg / ml to about 1000 mg / ml, about 300 mg / ml to about 1000 mg / ml, about 400 mg / ml to about 1000 mg / ml, about 400 mg / ml to about 800 mg / ml, about 400 mg / ml to about 700 mg / ml, about 400 mg / ml to about 600 mg / ml, etc.). In some embodiments, the composition (e.g., suspension) comprises particles containing a therapeutic biological agent (e.g., protein, hormone, antibody such as hIgG) and one or more flocculants in a concentration of about 50 mg / ml to about 1000 mg / ml (e.g., about 100 mg / ml to about 1000 mg / ml, about 200 mg / ml to about 1000 mg / ml, about 300 mg / ml to about 1000 mg / ml, about 400 mg / ml to about 1000 mg / ml, about 400 mg / ml to about 800 mg / ml, about 400 mg / ml to about 700 mg / ml, about 400 mg / ml to about 600 mg / ml, etc.).

[0160] In some embodiments, the composition (e.g., suspension) contains at least about 300 mg / ml (e.g., about 300 mg / ml to about 1000 mg / ml, about 400 mg / ml to about 1000 mg / ml, about 300 mg / ml to about 900 mg / ml, about 300 mg / ml to about 700 mg / ml, etc.) of protein particles. In some embodiments, the composition (e.g., suspension) contains at least about 300 mg / ml (e.g., about 300 mg / ml to about 1000 mg / ml, about 400 mg / ml to about 1000 mg / ml, about 300 mg / ml to about 900 mg / ml, about 300 mg / ml to about 700 mg / ml, etc.) of protein particles and a flocculant. In some embodiments, the composition (e.g., suspension) contains particles containing antibody in an amount of at least about 300 mg / ml (e.g., about 300 mg / ml to about 1000 mg / ml, about 400 mg / ml to about 1000 mg / ml, about 300 mg / ml to about 900 mg / ml, about 300 mg / ml to about 700 mg / ml, etc.). In some embodiments, the composition (e.g., suspension) contains particles containing antibody and a flocculant in an amount of at least about 300 mg / ml (e.g., about 300 mg / ml to about 1000 mg / ml, about 400 mg / ml to about 1000 mg / ml, about 300 mg / ml to about 900 mg / ml, about 300 mg / ml to about 700 mg / ml, etc.). In some embodiments, the antibody is hIgG. In some embodiments, the flocculant includes PS80, PEG (e.g., PEG300, PEG600, etc.), propylene glycol, or a combination thereof. In some embodiments, the flocculant comprises PS80. In some embodiments, the flocculant comprises PS80 and PEG. In some embodiments, the flocculant is a 1:1 mixture of PS80 and PEG. In some embodiments, the flocculant comprises PS80 and propylene glycol. In some embodiments, the flocculant is a 1:1 mixture of PS80 and propylene glycol.

[0161] The ratio of two anti-cancer monoclonal antibodies (trastuzumab and rituximab) when co-formulated in microparticles can be maintained by treatment, as measured by CEX. In embodiments having an aqueous formulation (supply) containing 48% rituximab and 52% trastuzumab, a similar or identical ratio may be present in the final microparticle suspension formulation, ensuring appropriate doses of both therapeutic agents.

[0162] Excipients may be selected to maintain stability. In some embodiments, arginine hydrochloride (amino acid), sucrose (carbohydrate), and PS80 (surfactant) were able to maintain the stability of the sample through microparticle formation and when stored at 40°C for one month. In some embodiments, samples with and without hyaluronidase enzyme in the microparticles may exhibit similar protein quality attributes, and the effect of hyaluronidase on the quality of the therapeutic protein is minimal.

[0163] In some embodiments, the chemical stability of the hyaluronidase enzyme in microparticles was achieved by selecting the correct excipients. In some embodiments, the stability of enzyme activity in microparticles containing rituximab and hyaluronidase can be maintained. Excipients such as sucrose (carbohydrate) and PS80 (surfactant) maintain the stability of the hyaluronidase enzyme through microparticle formation and when stored at 40°C for one month.

[0164] In some embodiments, samples of rituximab microparticles, with and without hyaluronidase enzyme in the microparticles, exhibited similar protein quality attributes regardless of the carrier liquid in which they were suspended. The stability of the formulations over one month of storage at 40°C may also be similar.

[0165] This disclosure provides compositions and methods for subcutaneous administration of particle suspensions of therapeutic biological agents and hyaluronan degraders to treat a disease or condition in a subject requiring treatment of that disease or condition. This specification provides a method for treating a disease or condition in a subject requiring treatment of that disease or condition, comprising administering to the subject a pharmaceutically effective amount of a composition comprising a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein substantially all of the particles comprise at least one therapeutic biological agent or a salt thereof and a hyaluronan degrader. In some embodiments, the disease or condition is cancer, an inflammatory disease, or an immune disease. In certain embodiments, the particle suspension of therapeutic biological agents and hyaluronan degraders increases the bioavailability of the subcutaneously administered therapeutic biological agent. As disclosed herein, particles comprising hyaluronan degraders can increase the bioavailability of a subcutaneously administered therapeutic biological agent by more than about 20%. See Figure 16.

[0166] When therapeutic compositions containing therapeutic biological agents, e.g., mAbs, and hyaluronane degraders, e.g., hyaluronidase, are clinically applied, it is generally beneficial to prepare a pharmaceutical or therapeutic composition that is appropriate for the intended use. In certain embodiments, the pharmaceutical composition may contain, for example, at least about 0.1% of the active therapeutic biological agent. In some embodiments, the active therapeutic biological agent may constitute about 2% to about 99% or about 50% to about 99% of the unit weight, and may include, for example, any range that can be derived therefrom. In some embodiments, the concentration of the hyaluronane degrader in the composition is less than about 10 mg / mL.

[0167] As described herein, CD20 (also known as Bp35) is a B lymphocyte-restrictive differentiation antigen that is expressed during early pre-B cell development and persists until plasma cell differentiation. Because this antigen is expressed at very high densities on the surface of malignant B cells, i.e., B cells whose unsustainable proliferation can lead to B-cell lymphoma, CD20 may be a useful target for B-cell lymphoma. The Food and Drug Administration (FDA) has approved the therapeutic use of the anti-CD20 antibody, rituximab (RITUXAN®), for use in relapsed and previously treated low-grade non-Hodgkin lymphoma (NHL). Rituximab acts by binding to the CD20 antigen on B cells, thereby causing B cell lysis through a mechanism thought to involve complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). In certain embodiments, the therapeutic biological agent is an antibody. In certain embodiments, the antibody is an anti-CD20 antibody.

[0168] This specification provides compositions and methods for treating a disease or condition in subjects requiring treatment of the disease or condition, the methods comprising administering to the subject a pharmaceutically effective amount of the composition, comprising a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein each particle (e.g., substantially all of the particles) comprises at least one therapeutic biological agent or a salt thereof and a hyaluronan degrader, and the concentration of the therapeutic biological agent or a salt in the composition is greater than about 250 mg / mL. In some embodiments, this specification provides methods for treating cancer, inflammatory diseases, or immune diseases in subjects requiring treatment of cancer, inflammatory diseases, or immune diseases, the methods comprising administering to the subject a pharmaceutically effective amount of the composition, comprising a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein each particle (e.g., substantially all of the particles) comprises at least one therapeutic biological agent or a salt thereof and a hyaluronan degrader, and the concentration of the therapeutic biological agent or a salt in the composition is greater than about 250 mg / mL. Furthermore, this specification provides a method for administering a pharmaceutically effective composition comprising a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein substantially all of the particles comprise at least one therapeutic biological agent or a salt thereof and a hyaluronan degrader, and the concentration of the therapeutic biological agent or a salt thereof in the composition is greater than about 250 mg / mL. In certain embodiments, the particles comprise at least two therapeutic biological agents and a hyaluronan degrader. In some embodiments, the particles comprise at least two antibodies and a hyaluronan degrader. In certain embodiments, the particles comprise at least two monoclonal antibodies and a hyaluronan degrader.

[0169] The terms “to treat” or “to treat” or “treatment” generally refer to therapeutic measures, such as treating, reversing, and / or downregulating a disease or condition. As used herein, “to treat” or “to treat” means administering a therapeutic biological product, such as a composition containing either an antibody or an antigen-binding fragment of the herein, internally or externally to a subject having, or suspected of having, one or more symptoms of a disease for which the biological product has therapeutic or prophylactic activity. Typically, therapeutic biological products and hyaluronane degraders are administered in an amount effective to alleviate one or more disease symptoms in the treated subject or population, whether by inducing regression of such symptoms or by inhibiting their progression to any clinically measurable extent. The amount of therapeutic biological product effective to alleviate any particular disease symptom may vary depending on factors such as the subject's or patient's condition, age, and weight, as well as the therapeutic biological product's ability to induce a desired response in the subject or patient. Whether disease symptoms are alleviated can be assessed by any clinical measurement typically used by a physician or other skilled healthcare provider to assess the severity or progression of the symptoms. The term further includes delaying the onset of symptoms associated with the disorder and / or reducing the severity of symptoms of such disorder. The term further includes improvement of existing uncontrolled or undesirable symptoms, prevention of additional symptoms, and improvement or prevention of the underlying causes of such symptoms. Thus, the term indicates that a beneficial outcome has been given to a human or animal subject having a disorder, disease, or symptoms, or being at risk of developing such disorder, disease, or symptoms. As used herein, “treatment” means therapeutic treatment and diagnostic use when applied to human or veterinary subjects. “Treatment” includes contact of the therapeutic biological products of the present disclosure, e.g., antibodies or antigen-binding fragments, to a human or animal subject when applied to human or veterinary subjects. Subjects requiring treatment for cancer include those already having benign, precancerous, or non-metastatic tumors, and those for whom the development or recurrence of cancer should be prevented.In some embodiments, the treatment or the objective or outcome of the treatment may be to reduce the number of cancer cells, reduce the size of the primary tumor, inhibit (i.e., slow down, preferably stop) cancer cell invasion into peripheral organs, inhibit (i.e., slow down, preferably stop) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more of the symptoms associated with the disorder. In some embodiments, the effectiveness of the treatment may be measured by assessing the time of survival, time to disease progression, response rate (RR), duration of response, and / or quality of life. As used herein, the terms “prevent” or “prevention” refer to any action that inhibits or delays the onset of a disease or condition in a subject that requires inhibition or delay of the onset of the disease or condition, including administering to the subject a pharmaceutically effective amount of the composition of this disclosure. In certain embodiments of the present disclosure, “treating” a subject suffering from a disease or condition includes, but is not limited to, (i) slowing, stopping or reversing the progression of the disease or condition; (ii) slowing, stopping or reversing the progression of the symptoms of the disease or condition; (iii) reducing the likelihood of recurrence of the disease or condition; and / or (iv) reducing the likelihood of recurrence of the symptoms of the disease or condition.

[0170] Non-limiting examples of cancers that can be treated by the compositions and methods described herein include, but are not limited to, tumors of the gastrointestinal tract (colon cancer, rectal cancer, colorectal carcinoma, colorectal cancer, colorectal adenoma, hereditary nonpolyposis type 1, hereditary nonpolyposis type 2, hereditary nonpolyposis type 3, hereditary nonpolyposis type 6; colorectal cancer, hereditary nonpolyposis type 7, small intestine and / or large intestine cancer, esophageal cancer, callous formation with esophageal cancer, gastric carcinoma, pancreatic cancer, pancreatic endocrine tumors), endometrial cancer, dermatofibrosarcoma protuberans, gallbladder cancer, bile duct tumors, prostate cancer, prostate Adenocarcinoma, kidney cancer (e.g., Wilms tumor type 2 or 1), liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma, hepatocellular carcinoma), bladder cancer, embryonal rhabdomyosarcoma, germ cell tumors, trophoblastic tumors, testicular germ cell tumors, ovarian, uterine, premature teratoma of the epithelial ovary, sacrococcygeal tumors, choriocarcinoma, placental trophoblastic tumors, epithelial adult tumors, ovarian cancer, serous ovarian cancer, ovarian cord tumors, cervical cancer, cervix cancer, small cell and non-small cell lung cancer, nasopharyngeal cancer, breast cancer (e.g., ductal carcinoma, invasive intraductal carcinoma, sporadic; breast cancer, morbidity of breast cancer, type 4 breast cancer, breast cancer-1, breast cancer-3);Breast-ovarian cancer), squamous cell carcinoma (e.g., in the head and neck), neurogenic tumors, astrocytoma, ganglioblastoma, neuroblastoma, lymphoma (e.g., Hodgkin's disease, non-Hodgkin lymphoma, B-cell, Burkitt, cutaneous T-cell, histiocytic, lymphoblastic, T-cell, thymic, B-cell non-Hodgkin lymphoma), glioma, adenocarcinoma, adrenal tumors, hereditary adrenocortical carcinoma, malignant tumors of the brain, various other cancers (e.g.) , bronchogenic large cell, glandular, malignant ascites, Ehrlich-Letre ascites, epidermoid, large cell, Lewis lung, medullary, mucoepidermal, oat cell, small cell, spindle cell, spindle cell, transitional cell, undifferentiated, carcinosarcoma, choriocarcinoma, cystadenocarcinoma), ependymoblastoma, epithelioma, erythroleukemia (e.g., Friend, lymphoblastoma), fibrosarcoma, giant cell tumor, glial tumor, glioblastoma (e.g., pleomorphic, astrocytoma), glioma hepatocyte Hepatoma), heterohybridoma, heteromyeloma, histiocytoma, hybridoma (e.g., B cell), adrenal tumor, insulinoma, islet tumor, keratoma, epithelioid leiomyoma, leiomyosarcoma, leukemia (e.g., acute lymphoblastic, acute lymphoblastic, acute lymphoblastic pre-B cell, acute lymphoblastic T cell leukemia, acute megakaryoblastic, monocytic, acute myeloid, acute myeloid, acute myeloid with eosinophilia, B cell, basophilic, chronic myeloid, chronic, B cell, eosinophilic, friend, granulocytic or myeloid, pilocytic cell, lymphocytic, megakaryoblastic, monocytic, monocytic-macrophage, myeloblastic, myeloid, myelomonocytic, plasma cell, pre-B cell, promyelocytic, subacute, T cell, lymphoid neoplasm, Predisposition to myeloid malignancies (acute nonlymphocytic leukemia), lymphosarcoma, melanoma, breast tumors, mast cell tumors, medulloblastoma, mesothelioma, metastatic tumors, monocytic tumors, multiple myeloma, myelodysplastic syndrome, myeloma, nephroblastoma, glial tumors of nerve tissue, neuronal tumors of nerve tissue, schwannoma, neuroblastoma, oligodendroglioma, osteochondroma, osteomyeloma, osteosarcoma (e.g., Ewing's), osteoporosis, bone metastases, papillomas, transitional cells, pheochromocytoma, pituitary tumors (invasive), plasmacytoma, retinoblastoma, rhabdomyosarcoma, sarcomas (e.g., Ewing's, histiocytic, Jensen's, osteogenic, reticular), schwannoma, subcutaneous tumors, teratomas (e.g., pluripotent), teratomas, testicular tumors, thymoma and follicular epithelioma, gastric cancer, fibrosarcoma, glioblastoma multiforme;This includes, but is not limited to, any solid or non-solid cancer and / or cancer metastases, including, multiple glomus tumors, Lie-Fraumeni syndrome, liposarcoma, Lynch's familial cancer syndrome type II, male germ cell tumors, mast cell leukemia, medullary thyroid, multiple meningiomas, endocrine neoplasia, myxosarcoma, paraganglioma, familial nonchromophilic tumor, pilomatoma, papillary, familial and sporadic, rhabdoid diathesis syndrome, familial, rhabdoid tumor, soft tissue sarcoma, or Turcott syndrome with glioblastoma.

[0171] In some embodiments, the therapeutic biological agent is an immunotherapy. In some embodiments, the immunotherapy is an anti-CD20 antibody. In certain embodiments, the anti-CD20 antibody is rituximab. In certain embodiments of the compositions and methods described herein, a treatment for non-Hodgkin lymphoma (NHL) in a subject requiring treatment for non-Hodgkin lymphoma (NHL) may be useful for treatment, comprising administering to the subject a pharmaceutically effective amount of a composition comprising a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein the particles (e.g., substantially all of the particles) comprise at least one therapeutic biological agent or a salt thereof and a hyaluronan degrader, and the concentration of the therapeutic biological agent or a salt thereof in the composition is greater than about 250 mg / mL. Any antibody capable of binding to the CD20 antigen as described herein may be used in the methods of this disclosure. Antibodies that bind to the CD20 antigen include, for example, C2B8 (rituximab; RITUXAN®) (U.S. Patent No. 5,736,137, expressly incorporated herein by reference), yttrium-

[90] -labeled 2138 mouse antibody named Y2B8 (U.S. Patent No. 5,736,137, expressly incorporated herein by reference), mouse IgG2a 131 (BEXXAR®) (U.S. Patent No. 5,595,721, expressly incorporated herein by reference) which is optionally labeled with 131 1 to produce 131 1-B1 antibody, mouse monoclonal antibody 1F5 (Press et al. Blood 69(2):584-591 (1987)), chimeric 2H7 antibody (U.S. Patent No. 5,677,180, expressly incorporated herein by reference), and International Leukocyte Typing Monoclonal antibodies available from the Workshop include L27, G28-2, 93-1 133, B--Cl, or NU--B2 (Valentine et al., In: Leukocyte Typing III (McMichael, Ed., p.440, Oxford University Press (1987))).

[0172] In certain embodiments of this disclosure, the anti-CD20 antibody is rituximab. Rituximab is a genetically engineered chimeric mouse / human monoclonal antibody. Rituximab is an IgG, kappa immunoglobulin containing mouse light chain and heavy chain variable region sequences, as well as a human constant region sequence. Rituximab has a binding affinity of approximately 8.0 nM for the CD20 antigen and is commercially available, for example, from Genentech (South San Francisco, CA).

[0173] In other embodiments, the antibody is trastuzumab (Herceptin; Genentech, San Francisco, CA). Trastuzumab is a humanized monoclonal antibody that binds to the extracellular portion of the HER2 ectodomain and prevents a cascade that leads to dimerization and growth factor expression. In certain embodiments, trastuzumab is used to treat breast cancer, esophageal cancer, gastric cancer, or other new or metastatic cancers that overexpress HER2.

[0174] Furthermore, this specification provides a method for treating an inflammatory disease in a subject requiring treatment for an inflammatory disease, comprising administering to the subject a pharmaceutically effective amount of a composition comprising a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein the particles (e.g., substantially all of the particles) comprise at least one therapeutic biological agent or a salt thereof and a hyaluronan degrader, and the concentration of the therapeutic biological agent or a salt thereof in the composition is greater than about 250 mg / mL.

[0175] In some embodiments of the methods described herein, inflammatory diseases include, but are not limited to, joint diseases, ophthalmic diseases, retinal diseases, Crohn's disease, irritable bowel syndrome, Sjögren's disease, tissue graft rejection, asthma, multiple sclerosis, scleroderma, Goodpasture syndrome, atherosclerosis, chronic idiopathic thrombocytopenic purpura, Addison's disease, Parkinson's disease, Alzheimer's disease, diabetes mellitus, septic shock, myasthenia gravis, inflammatory pelvic disease, inflammatory bowel disease, urethritis, uveitis, sinusitis, pneumonitis, encephalitis, meningitis, myocarditis, osteomyelitis, myositis, hepatitis, gastritis, enteritis, appendicitis, pancreatitis, or cholecystitis.

[0176] In some embodiments, the compositions and methods described herein are used to treat immunological diseases, such as secondary acquired hypogammaglobulinemia, chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, inflammatory myopathy, Lambert-Eaton myasthenic syndrome, multifocal motor neuropathy, myasthenia gravis, Mersch-Woltmann syndrome, secondary hypogammaglobulinemia-specific antibody deficiency, acute disseminated encephalomyelitis, autoimmune hemolytic anemia, and scarring pemphigoid. It is useful in treating acne, Evans syndrome, fetal maternal / neonatal alloimmune thrombocytopenia (FMAIT / NAIT), hemophagocytic syndrome, high-risk allogeneic hematopoietic stem cell transplantation, IgM-deficient protein neuropathy, kidney transplantation, multiple sclerosis, ocular clonus-myoclonus ataxia, post-transfusion purpura, toxic epidermal necrolysis / Stevens-Johnson syndrome (TEN / SJS), toxic shock syndrome, Alzheimer's disease, multiple myeloma, sepsis; B-cell tumors, trauma, or bacterial, viral, or fungal infections. In some embodiments, the immune disease is an autoimmune disease. In certain embodiments, autoimmune diseases include, but are not limited to, multiple sclerosis, scleroderma, type 1 diabetes, rheumatoid arthritis, thyroiditis, Raynaud's syndrome, Sjögren's syndrome, autoimmune uveitis, autoimmune myocarditis, inflammatory bowel disease, amyotrophic lateral sclerosis (ALS), systemic lupus, neuromyelitis optica, idiopathic thrombocytopenic purpura, myasthenia gravis, ulcerative colitis, Crohn's disease, polyarthritis, graft-versus-host reaction, juvenile-onset diabetes, Hashimoto's thyroiditis, Graves' disease, pernicious anemia, chronic active (lupoid) hepatitis, psoriatic arthritis, or neurodermatitis. "To decrease" or "to reduce" means to become less or smaller in number, amount, size, or intensity. In certain embodiments, reducing the risk of a disease (e.g., focal segmental glomerulosclerosis (FSGS)) includes reducing the likelihood of developing the disease by at least about 20%, e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, reducing the risk of a disease includes delaying the onset of the disease, e.g., at least about 6 months, e.g., about 1 year, e.g., about 2 years, about 5 years, or about 10 years.

[0177] As described herein, particle-based compositions having a flocculant can directly address the current challenges of subcutaneous administration of therapeutic biological agents, such as mAbs, and hyaluronane degraders, such as hyaluronidase, by enabling injection with low injection force without requiring resuspension before injection. In some embodiments, the particles and flocculant are suspended in a non-aqueous liquid carrier and remain substantially suspended in the liquid carrier for at least one month, eliminating the need for complex and time-consuming resuspension procedures. The compositions described herein can be used in pre-filled syringes or pre-filled portable drug delivery injection devices. The highly dispersible nature of the particles in the compositions described herein enables patient-friendly subcutaneous injection. In the subcutaneous space, as described herein, the therapeutic biological agent contained in the particles readily returns to its original monomeric state upon injection, enabling full bioavailability. In certain embodiments, the composition achieves a higher content without impairing the quality of the therapeutic biological agent, and thus enables the easy delivery of the therapeutic biological agent, e.g., mAb, and hyaluronan degrading agent, e.g., hyaluronidase, by subcutaneous injection, thereby treating the disease or condition in subjects requiring treatment of the disease or condition, where the disease or condition is cancer, an inflammatory disease, or an immune disorder. In some embodiments, the disease or condition is cancer. In some embodiments, the disease or condition is an inflammatory disease or condition. In certain embodiments, the disease or condition is an immune disorder.

[0178] The pharmaceutical compositions and methods disclosed herein can be administered to a subject by any preferred route of administration, including, for example, parenteral administration. In some embodiments, the compositions are administered by subcutaneous injection. The term “pharmaceutical composition” as disclosed herein means a preparation that, for example, is in a form that enables the therapeutic biological agent contained in the composition when administered to a subject, and does not contain any additional components that would make the composition unacceptably toxic to the subject to which it is administered. Such compositions are sterile. A “sterile” composition or preparation is sterile or free from all living microorganisms and their spores.

[0179] The terms “injectability” or “syringeability” refer to the relative ease with which the liquid compositions described herein can be administered to a subject through the use of an injection device, e.g., a syringe or a portable drug delivery injection device. In particular, injectability is partially influenced by the viscosity of the composition, more importantly, the sedimentation of particles, the injection or transfer flow rate, and the characteristics of the needle, e.g., length and gauge. In some embodiments, injectability is determined by measuring the viscosity of the composition at various shear rates. In some embodiments, injectability is determined by measuring the release force and / or sliding force required to operate an injection device consisting of a barrel, plunger, and needle. In certain embodiments, the barrel of the syringe has an inner diameter of at least 6 mm. As described herein, the injectability of a composition comprising multiple particles including a flocculant and at least one therapeutic biological agent and a hyaluronan degrader is better than that of an aqueous co-formulation having substantially the same concentration of aqueous monomer therapeutic biological agents. The terms “injectability” or “syringeability” can also refer to the injectability of a pharmaceutical composition through a syringe equipped with a 16-33 gauge needle, optionally a thin-walled or ultra-thin-walled (UTW) needle. In certain embodiments, the syringe is equipped with a needle that is at least 8 mm in length. Injectability depends on factors such as the pressure or force required for injection, the uniformity of the flow, the quality of aspiration, and the absence of clogging. As described herein, injectability can be evaluated by comparing the injection force of a composition after a period of time to a standard particulate composition without the addition of a flocculant. The compositions described herein can improve injectability after a period of time, and the injection force is reduced by at least 10%, preferably at least 30%, more preferably at least 50%, and most preferably at least 75%, compared to a standard particulate composition having the same concentration of therapeutic biological agent under different same conditions.Alternatively, the injectability of a composition can be evaluated by comparing the time or injection force required to inject a coagulated volume, e.g., about 2.0 mL, preferably about 1.5 mL, more preferably about 1.0 mL, and most preferably about 0.5 mL, of the composition, when the syringe is pressed with the same force over a period of time, for example, with particle sedimentation over a period of time. The term "flow rate" refers to the volume of liquid composition that can pass through a given cross-sectional area per unit time. Generally, the flow rate formula is Q = A × v, where Q is the flow rate, A is the cross-sectional area at a point in the flow path, and v is the average velocity of the liquid at that point. In certain embodiments, the flow rate is constant. In certain embodiments, the flow rate is at least about 0.1 mL / second.

[0180] The term "injection release force" refers to the force required to overcome the friction between the syringe barrel and plunger of a standard injection device before the contents of the syringe can be discharged at a steady rate, for example, the maximum force required to break the static friction of the plunger. The force is applied at the end facing outward on the syringe plunger shaft and directed along the axis of the syringe barrel. The contents of the syringe are discharged through a syringe needle of a predetermined gauge and length. In certain embodiments, the injection release force is measured by a load cell positioned at the end facing outward on the syringe plunger during operation.

[0181] The terms “syringe extrusion force,” “injection force,” “injection gliding force,” or “gliding force” are used interchangeably herein and refer to the force required to maintain the stable discharge of the contents of a standard injection device, e.g., the force required to maintain the movement of the plunger once static friction is overcome. The force is applied at the end of the syringe plunger shaft facing outward and directed along the axis of the syringe barrel. The contents of the syringe are discharged through a syringe needle of a predetermined gauge and length. The term “Newtonian region” or “N” means a range of shear stress that is linearly or substantially linearly proportional to the local strain rate at all points. In some embodiments, the addition of a flocculant to the particle suspension reduces the syringe extrusion force.

[0182] The administration of the compositions described herein may include administration using an 18-33 gauge needle. An 18-33 gauge needle may have a length of about 19 mm (3 / 4 inch) or less, or preferably about 13 mm (1 / 2 inch) or less. In some embodiments, the administration of the compositions uses a 27-33 gauge needle. In some embodiments, a 27-33 gauge needle has a length of about 13 mm (1 / 2 inch) or less. In certain embodiments, the compositions are dispensed from needles having gauges in the 18-33 gauge range, for example, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32-33 gauge. In some embodiments, the compositions are dispensed from needles having gauges in the 27-30 gauge range. In certain embodiments, the compositions are dispensed from needles having gauges in the 25-27 gauge range. In certain embodiments, the compositions are dispensed from needles having a 27 gauge.

[0183] The length of the needle used may affect the required syringe extrusion force and the time required to dispense the composition. As shown below in Example 9, the length of the needle used may affect the syringe extrusion force. In some embodiments, the needle may be up to 8 mm in length. In some embodiments, the needle may be up to 13 mm in length. The needle may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mm in length. The needle may be 8 to 13 mm in length without departing from the scope of the disclosure. In some embodiments, the needle length may be between 1 to 2 mm, 2 to 3 mm, 3 to 4 mm, 4 to 5 mm, 5 to 6 mm, 6 to 7 mm, 7 to 8 mm, 8 to 9 mm, 9 to 10 mm, 10 to 11 mm, 11 to 12 mm, 12 to 13 mm, 13 to 14 mm, or 14 to 15 mm without departing from the scope of the disclosure. In some embodiments, the needle may be 6–8 mm or 10–12 mm in length, without departing from the scope of the present disclosure. A needle having a length of about 8 mm may take about 6 seconds to inject 2 mL of suspension. A needle having a length of about 12 mm may take about 11 seconds to inject 2 mL of suspension. A needle having a length of about 12 mm may take about 11 seconds to inject 2 mL of suspension.

[0184] In some embodiments, the composition is distributed using an injection force of less than about 70 N, for example, about 60, 50, 40, 30, 25, 20, 15, 10, or 5 N. In some embodiments, the composition is distributed using an injection force of less than about 25 N. In certain embodiments, the composition is distributed using an injection force of less than about 20 N. In certain embodiments, the composition is distributed using an injection force of less than about 15 N. In some embodiments, the composition is distributed using an injection force of less than about 10 N. In certain embodiments, the composition is distributed using an injection force of less than about 5 N. In some embodiments, the injection force increases at a rate lower than the viscosity of the composition as the concentration of the therapeutic biological agent in the composition increases. In certain embodiments, the injection force remains substantially the same for at least one month under container-closed storage conditions at less than about 40°C.

[0185] In other cases, the compositions comprising the plurality of particles and flocculants described herein further optionally include administering at least one hyaluronane degrading agent, such as hyaluronidase, which is administered simultaneously with the composition, sequentially, or intermittently. In some embodiments, the administered composition is less than about 20.0 mL, for example, 15.0, 10.0, 5.0, 2.0, 1.5, 1.0, or 0.5 mL. In some embodiments, the administered composition is less than about 2.0 mL. In certain embodiments, the administered composition is less than about 1.5 mL. In some embodiments, the administered composition is less than about 1.0 mL. In certain embodiments, the administered composition is less than about 0.5 mL.

[0186] In some embodiments, the composition dissolves in less than approximately 10, 9, 8, 7, 6, 5, 4, 3, 2, and 1 minute after administration. In certain embodiments, the composition dissolves in less than approximately 60 seconds after administration, for example, in 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, ... 10, 9, 8, 7, 6, 5, 4, 3, 2, and 1 seconds. In certain embodiments, the composition dissolves immediately after administration.

[0187] The compositions described herein can be used for parenteral administration and are formulated for injection via subcutaneous routes, for example, without requiring manual stirring or pre-mixing of the injection device before use. Pharmaceutical compositions suitable for injectable use contain a sterile non-aqueous liquid carrier containing sterile particles. In all cases, the composition must be sterile and fluid enough to be readily injectable. The pharmaceutical composition must also be stable under current Good Manufacturing Practices (cGMP) and storage conditions.

[0188] This disclosure generally relates to compositions for use in syringes or portable drug delivery injection devices. In certain embodiments, the composition is contained within a syringe or portable drug delivery injection device. For example, the syringes disclosed herein may be pre-filled syringes. In certain embodiments, the syringe or portable drug delivery injection device is pre-filled with the composition. Examples of portable drug delivery injection devices include pen syringes or autoinjectors (e.g., autoinjectors, patches). The compositions described herein may be sealed in disposable syringes, cartridges, or any other container closures made of glass or plastic. In some embodiments, the composition is administered by syringe injection. In some embodiments, the composition is dispensed from a pre-filled syringe. In certain embodiments, the portable drug delivery injection device is configured to automatically or semi-automatically deliver the drug to the patient using a wireless communication system.

[0189] Automated injectors, such as auto-injectors, pen syringes, or drug pens, are pre-filled, pre-assembled injection devices that enable parenteral administration of therapeutic drugs. The advantage of automated injectors is that they contain a measured dose of therapeutic drug in a sealed, sterile cartridge or injection device. Automated injectors are useful for people with low dexterity, poor eyesight, or those who require portability to administer therapeutic drugs on a timely basis, and can also reduce fear or resistance to self-injecting therapeutic drugs, which increases the likelihood of a person using their medication. Automated injectors are most useful in emergencies, as they allow for quick and easy self-administration of therapeutic drugs without the need to measure dosages.

[0190] Ensuring complete delivery of particle suspensions of therapeutic biological agents in the compositions and methods disclosed herein is often important for particle suspensions, where the injection force can already be relatively high. At high shear, as is often the case with portable drug delivery injection devices, the problem of ensuring complete injection of a unit dose at high injection forces can be complicated by particle sedimentation in the composition, making user operation difficult.

[0191] In automated injectors, the particles of therapeutic biological agents in the composition are likely to be stored as a particle suspension for subsequent injection. However, long-term storage of particle suspensions (e.g., 3 to 12 months) has drawbacks, such as particle sedimentation, which can lead to high injection force. These automated injectors require the user to manually shake the syringe body to facilitate resuspending of particles immediately before injection. Unfortunately, steps such as manually shaking the automated injector increase the time required to administer a certain dose of therapeutic drug, which is undesirable in many emergency medical situations where rapid delivery of therapeutic drug is required.

[0192] In some embodiments, the composition is administered by syringe or portable drug delivery injection device. In certain embodiments, the composition is contained within the syringe or portable drug delivery injection device. In certain embodiments, the composition is administered by syringe. In certain embodiments, the composition is administered by portable drug delivery injection device. In certain embodiments, the portable drug delivery injection device is a pen syringe. In some embodiments, the portable drug delivery injection device is an autoinjector. In certain embodiments, the syringe or portable drug delivery injection device is pre-filled with the composition. In some embodiments, the composition is administered in one or more doses. In some embodiments, the composition is administered in a single dose. In certain embodiments, the composition is administered in multiple doses.

[0193] In some embodiments, the composition is administered by syringe. In some embodiments, the composition is administered by pen syringe. In certain embodiments, the composition is administered by autoinjector. In certain embodiments, the syringe, pen syringe, or autoinjector is pre-filled.

[0194] The compositions of this disclosure may be used to treat a disease or condition in subjects requiring treatment of that disease or condition. In some embodiments, the disease or condition is cancer. In some embodiments, the disease or condition is an inflammatory disease or condition. In certain embodiments, the disease or condition is an immune disease. In certain embodiments, the subject is a mammal, e.g., human or animal. The compositions described herein may be administered in vivo to a mammal, e.g., human or animal subject using a variety of known routes and techniques. For example, the compositions may be provided as an injectable suspension and administered by subcutaneous injection using a conventional needle and syringe or a portable drug delivery injection device. In certain embodiments, the compositions are administered to mammals. In certain embodiments, the compositions are administered to humans.

[0195] In certain embodiments of this disclosure, complete dissolution of the composition may occur immediately or within seconds upon administration, mitigating any immunological risks posed by particles persisting in the subcutaneous space. For example, administration of particles containing an mAb and hyaluronidase has been shown to produce a similar or improved pharmacokinetic (PK) profile (AUC, Cmax, and Tmax) compared to particles containing an equal dose of an mAb. In certain embodiments, the composition dissolves immediately after administration. In some embodiments, the composition has improved pharmacokinetics (PK) compared to a composition containing multiple particles, each containing at least one therapeutic biological agent.

[0196] The compositions described herein exhibit subcutaneous delivery of high concentrations of therapeutic biological formulations (300-750 mg / mL) without loss of biological activity. This has been achieved for various therapeutic biological formulations by using organic solvents, such as sesame oil, medium-chain triglycerides (MCTs), propylene glycol diesters (PGDs) of saturated vegetable fatty acids C8 and C10, and fatty acid esters (ethyl oleate (EO)), as liquid carriers. For example, ethyl oleate is a fatty acid ester with a viscosity of about 6 mPa·s at 25°C and does not chemically interact with particles during storage or in vivo dissolution.

[0197] In some embodiments, administration of particles containing a therapeutic biological agent and hyaluronidase, when administered by subcutaneous syringe injection, allows for increased bioavailability and fluid dispersion of the composition having a flocculant. As disclosed herein, the bioavailability of a composition having a flocculant, when administered subcutaneously without resuspension, can be significantly increased by particles containing a therapeutic biological agent and hyaluronidase. These hyaluronidase injection sites showed an improved rate of injection of the composition, as determined by the appearance of skin vesicles formed at the injection site.

[0198] Specific embodiments of this disclosure are described herein. Naturally, variations, alterations, modifications, and substitutions of the equivalents of these specific embodiments will be apparent to those skilled in the art by reading the foregoing description. The inventors expect that those skilled in the art will use such variations, alterations, modifications, and substitutions of equivalents as appropriate, and the inventors intend that this disclosure will be carried out in a manner different from that specifically described herein. Those skilled in the art will readily recognize various non-essential parameters that can be altered, changed, or modified to produce essentially similar results. Accordingly, this disclosure includes all variations and equivalents of the subject matter enumerated in the claims appended herein, as permitted by applicable law. Furthermore, any combination of the elements described above in all possible variations thereof is encompassed herein unless otherwise indicated herein or unless it is clearly inconsistent with the context.

[0199] The general nature of this disclosure will be more readily understood by referring to the following examples, which are included solely for illustrative purposes of certain aspects and embodiments of the disclosure and are not intended to limit it.

[0200] kit In various embodiments, a kit is envisioned comprising a pharmaceutically effective composition comprising a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein substantially all of the particles comprise at least one therapeutic biological agent or a salt thereof and a hyaluronan degrader. For example, the kit may comprise one or more additional containers, each containing one or more of various materials (e.g., reagents and / or devices in optionally concentrated forms) that are commercially and user-desirable for use of the compositions described herein. Non-limiting examples of such materials include, but are not limited to, diluents, filters, needles, syringes, cartridges, devices, carriers, packages, containers, contents and / or vial and / or tube labels listing instructions for use, as well as accompanying documentation with instructions for use. In some embodiments, this embodiment contemplates a kit for administering the therapy of the embodiment. The kit may comprise one or more sealed, pre-filled syringes, cartridges, or portable drug delivery injection devices containing any of the pharmaceutically effective compositions of this disclosure. The kit may include, for example, at least one therapeutic biological agent and a hyaluronan degrader, at least several particles, as well as reagents for preparing, formulating, and / or administering the components of the embodiment, or for performing one or more steps of the disclosed therapeutic method. In some embodiments, the kit may also include a suitable container that does not react with the components of the kit, such as an Eppendorf tube, syringe, bottle, tubing, or portable drug delivery injection device. The container may be made from a sterilizable material, such as plastic or glass. In certain embodiments, the composition is dispensed from a pre-filled syringe or portable drug delivery injection device.In a particular embodiment, the kit comprises a syringe or portable drug delivery injection device and a composition comprising a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein the particles (e.g., substantially all of the particles) comprise at least one therapeutic biological agent or a salt thereof and a hyaluronan degrader, and the concentration of the therapeutic biological agent or salt thereof in the composition is greater than about 250 mg / mL.

[0201] The kit may further include an instruction sheet outlining the procedural steps of the method described herein, which may follow substantially the same procedure as described herein or be known to those skilled in the art. The instruction information may be in a computer-readable medium containing machine-readable instructions that, when executed using a computer, result in the display of an actual or hypothetical procedure for delivering a pharmaceutically effective amount of a therapeutic biological product.

[0202] Labels may optionally be on or attached to the container. For example, if the letters, numbers, or other symbols forming the label are attached, stamped, or etched onto the container itself, the label is on the container; if, for example, it is present in a receiver or carrier that also holds the container, such as as accompanying documentation, the label is attached to the container. In addition, labels may be used to indicate that the contents are to be used for a particular therapeutic purpose. In addition, labels may indicate, for example, instructions for the use of the contents in the manner described herein. In certain embodiments, the pharmaceutical composition is contained in a pack or dispenser device containing one or more unit dosage forms containing the therapeutic biological products provided herein. For example, the pack contains metal or plastic foil, for example, a blister pack. Alternatively, the pack or dispenser device may be accompanied by instructions for administration. Alternatively, the pack or dispenser may be accompanied by a notice associated with the container in a form prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceuticals, and this notice reflects agency approval of the form of the drug for human or veterinary administration. Such notices are, for example, labeling approved by the U.S. Food and Drug Administration for prescription drugs, or approved package inserts. In some embodiments, a composition of multiple particles comprising the therapeutic biological agents and hyaluronan degraders provided herein, formulated in a suitable pharmaceutically liquid carrier, is prepared, placed in a suitable container, and labeled for the treatment of the indicated condition.

[0203] In some cases, the liquid compositions provided herein are formulated into pre-filled injection devices, such as syringes or portable drug delivery injection devices. In some embodiments, the particle suspension, for example, the composition, is formulated in an administered volume of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or about 2.5 mL. In some embodiments, the administered volume of the composition is less than about 2.5 mL. In certain embodiments, the administered volume of the composition is less than about 2.0 mL, preferably less than about 1.5 mL, more preferably less than about 1.0 mL, and most preferably less than about 0.5 mL.

[0204] In some embodiments, such a kit includes a pre-filled injection device of the present disclosure, e.g., a syringe or a portable drug delivery injection device, in a blister pack. The blister pack itself may be sterile inside. In some embodiments, the pre-filled injection device of the present disclosure, e.g., a syringe or a portable drug delivery injection device, may be placed inside such a blister pack before undergoing sterilization, e.g., final sterilization.

[0205] In some embodiments, provided herein are kits comprising a syringe or portable drug delivery injection device and a composition comprising a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein substantially all of the particles comprise at least one therapeutic biological agent or a salt thereof and a hyaluronan degrader, and the concentration of the therapeutic biological agent or salt thereof in the composition is greater than about 250 mg / mL.

[0206] The general nature of this disclosure will be more readily understood by referring to the following examples, which are included solely for illustrative purposes of certain aspects and embodiments of the disclosure and are not intended to limit it.

[0207] Example Table 1-1 Table 1-2 Table 1-3

[0208] Material Human IgG (IRHUGGF-LY, >97%) and bovine IgG (IRBVGGF) were obtained from Innovative Research as powder or aqueous solution. Bovine serum albumin (BSA) and human serum albumin (HSA) were purchased from Sigma-Aldrich. Monoclonal antibodies (mAbs) were supplied and received as aqueous solutions. A biosimilar of Roche's rituximab was purchased from a vendor that provided the antibody in an aqueous composition as 10 mg / mL rituximab, 9 mg / mL sodium chloride, 7.35 mg / mL sodium citrate dihydrate, and 0.7 mg / mL polysorbate 80. A biosimilar of Roche's trastuzumab was purchased from Genscript Biotech Corporation, which provided the antibody in an aqueous composition. Recombinant human hyaluronidase was purchased from Creative Biomart, Shirley, NY (40 KU / mL or 2.4 U / uL). The custom-made “supply solution” compositions used to process the particles were produced by desalting the received formulation, followed by concentration and addition of the desired excipients, or by modification through direct buffer exchange. All excipients used in the particle compositions are those used in existing approved biological formulations for injection. Concentration columns were sourced from Millipore Sigma (Amicon® Ultra 15 mL filters for protein purification and concentration with a 10-50 kDa cutoff) and used, as needed, to (i) reach the desired monoclonal antibody concentration and (ii) exchange buffer / excipients before particle formation. In certain cases, Zeba desalting columns (THERMO FISHER SCIENTIFIC® 87773) were also used to remove salt from the solution. Typically, the ratio of residual salt to protein in the desalted solution (weight / weight) was less than 1%, as determined by conductivity measurements and / or elemental analysis. All excipients were purchased from Sigma-Aldrich and used as received.

[0209] method FLOWCAM®: Particle sizing was measured using the FLOWCAM® dynamic image analysis instrument. The sample was diluted to approximately 1 mg / mL in isopropanol and passed through a thin channel. Images of the particles were recorded and analyzed by size and shape (count weighting). Particle size can also be measured directly in the suspension using optical diffraction (Horiba Partica LA-960 laser scattering particle size distribution analyzer).

[0210] Image Analysis: Particle diameter and roundness were measured using ImageJ analysis on SEM images. The analysis was performed on images, for example, 500× or 1000×. The ImageJ Particle Analysis tool was run on the images to identify objects with roundness >0.8 and size >0.5 μm, each accompanied by an object contour. These contours were visually inspected for good fit. Misidentified particles were manually excluded, overlooked particles were manually added, and measured using the ImageJ Diameter tool. Selected microscopic images were chosen for further analysis based on resolution that provided (i) minimal particle overlap, (ii) good contrast between particles and background, and (iii) particle occupancy of at least 10 pixels. This allowed particles to be easily identified and reduced resolution-based errors. A binarization threshold was applied to separate particles from the background, and a watershed segmentation algorithm was applied to ensure that individual particles were measured separately. Next, the ImageJ tool "Analyze Particles" was applied to the binary image with the following parameters: roundness between 0.5 and 1.0; size between 5 and infinite square microns; exclusion of edges; and hole filling. The contours of the identified particles were superimposed onto the original image. Then, misidentified particles, such as clusters identified as single particles or particles whose contours did not match, were discarded. Missed particles were measured by manually tracking their contours and using ImageJ's measurement tools.

[0211] Accelerated storage protocol: All samples were transferred to glass-bottom plates and 2R Schott vials for aging (typically 2 mL or 4 mL in volume depending on the sample). The glass-bottom plates and 2R Schott vials were sealed with Parafilm and placed in an oven at 4°C, 25°C, 40°C, 50°C, or 60°C, and visually inspected throughout the aging period to ensure integrity and stability.

[0212] Viscosity Measurement: Unless otherwise specified, suspension viscosity was measured at 25°C using an AR-G2 rheometer (TA Instruments) and a 25mm plate. Measurements were taken over 1000 seconds. -1 The measurement was performed at a rate lower than the shear rate experienced with a 27-gauge needle. Each measurement was repeated three times (with an interval of approximately 60 seconds between repetitions) to assess the short-term physical stability of the suspension. Calibration standards were recorded before each measurement to verify instrument settings. Syringe dispensing force was measured using a pre-filled glass syringe with a fixed force sensor and 27G needle during the dispensing of a 0.5 ml suspension (500 mg / mL) at 0.1 ml / second (Schott syriQ BioPure®).

[0213] Injectability Measurement: Unless otherwise specified, syringe ejection force (injection force) was measured using a 1 mL Norm-ject model syringe with a custom-made force sensor and a 27-gauge ultrathin-walled needle (TSK) during the dispensing of 1 mL of suspension (400 mg / mL particles) at a rate of 0.1 mL / second.

[0214] Karl Fischer: The moisture content was tested using Karl Fischer analysis. Approximately 100 mg of particles were heated in an oven at 150°C, and the released water was determined electrochemically.

[0215] Skeleton density: Skeleton density was measured by the gas specific gravity bottle method. The gas used was nitrogen or other suitable gas, and the particle mass was 0.0413 g.

[0216] Particle lysis: Phosphate-buffered saline (PBS) was added to the dry particle sample to produce a final concentration of 30 mg / mL (particle mass / mL solution, 96-well plate). After a certain period, 10 μL aliquots were taken from the sample vial, and the absorbance at 280 nm was measured and recorded. For all samples, the concentration of Ab, e.g., mAb, was plotted against time.

[0217] Salt content: Salt content was recorded by measuring sodium content using inductively coupled plasma atomic emission spectroscopy (ICP-OES). Calibration curves were prepared using a sodium standard (ICPTRACECERT®, 1000 mg / L). Quality control was completed using a diluted standard solution with a sodium concentration of 100 ppm. Subsequently, a particulate sample (approximately 15 mg) dissolved in 2 vol% nitric acid (10 mL) was analyzed, yielding an intensity for sodium below the instrument's detection limit of approximately 0.5 ppm. This indicated a sodium content of less than 0.034 wt% and a total salt content of less than 0.1 wt% (assuming equal removal of sodium citrate and sodium chloride).

[0218] Size exclusion chromatography (SEC) measurement: 20 μL of sample (1 mg / mL) was injected into an AGILENT ADVANCEBIO® SEC column (300 mm × 2.7 μm, 300 Å column) at a flow rate of 1 mL / min for 15 minutes in SEC buffer (25 mM phosphate, 250 mM NaCl pH 6.8). Peak analysis was performed by automated integration using the following parameters: gradient sensitivity = 0.5, peak width = 0, height reject = 0, area reject = 0, shoulder off, area percentage reject = 0, standard tangent skim mode, advanced baseline correction, front peak skim height ratio = 0, tail peak skim height ratio = 0, peak-to-valley ratio = 0, and skim-to-valley ratio = 0. Alternatively, 20 μL of the sample (1 mg / mL) was injected into an AGILENT ADVANCEBIO® SEC column (300 mm × 2.7 μm, 300 Å column) at a flow rate of 1 mL / min for 15 minutes in SEC buffer (25 mM phosphate, 250 mM NaCl pH 6.8). Peak analysis was performed by automated integration using the following parameters: gradient sensitivity = 0.5, peak width = 0, height reject = 0, area reject = 0, shoulder off, area percentage reject = 0, standard tangent skim mode, advanced baseline correction, front peak skim height ratio = 0, tail peak skim height ratio = 0, peak-to-valley ratio = 0, and skim-valley ratio = 0.

[0219] Cation exchange chromatography (CIEX) analysis: Charge variant analysis was performed on each sample at days 0, 7, and 30 under accelerated storage conditions using an AGILENT BIOMAB® NP5, 4.6 × 250 mm, PEEK ion exchange column. Samples were prepared at a concentration of 1 mg / mL after overnight dialysis in water. Buffer A was prepared with 30 mM phosphate, pH: 6.3, and NaCl: 0 mM. Buffer B was prepared as follows: Buffer A: 30 mM phosphate, pH: 6.3, and NaCl: 175 mM. Samples were run on a gradient set to start with 100% Buffer A, slope to 100% Buffer B over a period of 20 minutes, and then return the gradient to 100% Buffer A and 0% Buffer B over the next 1 minute. After re-equilibriumating the system in 100% Buffer A for 10 minutes, the next sample was injected. The integration was performed in manual skim peak mode, reflecting AGILENT® data.

[0220] Flow cytometry: 1 million Raji cells (100 μL per well) were plated per well in a 96-well "V-bottom" plate, and 10 μL of mAb, label, particles, or suspension was added to each well at a starting concentration of 200 μL. The dilution factor for mAb label, particles, and suspension was 3X. The plates were incubated at 4°C for 30 minutes. The plates were centrifuged at 2000 rpm for 5 minutes and washed three times with PBS. 100 μL of PE-conjugated goat anti-human IgG was added as a secondary antibody at a 1:200 dilution. The plates were centrifuged at 2000 rpm for 5 minutes and washed three times with PBS. The cells were then resuspended in 200 μL of cold PBS for analysis on a Life Technologies ATTUNE® NXT flow cytometer.

[0221] Scanning electron microscopy (SEM): Electron micrographs were collected from selected samples using either a HITACHI™ TM3030Plus or TM1000 benchtop microscope. Samples were immobilized on conductive tape and examined in a low-vacuum anti-static environment, eliminating the need for sample preparation.

[0222] Image Analysis: Selected microscope images were chosen for further analysis based on (i) minimal particle overlap, (ii) good contrast between particles and background, and (iii) resolution providing at least 10 pixels of particle occupancy. This allowed for easy identification of particles and reduced resolution-based errors. A binarization threshold was applied to separate particles from the background, and a watershed segmentation algorithm was applied to ensure that individual particles were measured separately. The ImageJ tool "Analyze Particles" was then applied to the binarized images with the following parameters: roundness between 0.5 and 1.0; size between 5 and infinite square microns; exclusion of edge particles; and hole filling. The contours of the identified particles were superimposed onto the original images. Misidentified particles, such as single particles or clusters identified as particles whose contours did not match, were then discarded. Missed particles were measured by manually tracking the particle contours and using ImageJ's measurement tools.

[0223] Density analysis: The particle skeleton density from the selected samples was determined by examining approximately 0.1 g of powder using the ACCUPYC® II 1340 gas-purged specific gravity bottle method.

[0224] Water content analysis: The water content in particles from selected samples was determined by heating approximately 0.1 g of powder in an oven equipped with a Karl Fischer titrator.

[0225] ELISA assay: An ELISA assay was used on selected samples to detect human antibodies in a denaturation-sensitive manner. Human IgG was first plated in PBS for 1 hour, followed by three washes of 4 minutes each in wash buffer (PBS + 0.05% Tween20), followed by blockage with 2% BSA (Sigma) in wash buffer for 45 minutes, followed by incubation with diluted (20 μg / mL) protein A-HRP (ABCAM®) for 45 minutes, followed by three washes of 3 minutes each in wash buffer, followed by incubation with TMB (ABCAM®) for 10 minutes, and finally quenched with stop solution (ABCAM®). Colorimetric readings were performed on a THERMO MULTISKAN SPECTRUM®.

[0226] Sub-Visible Particle (SvP) Analysis: Sub-visible particles (SvP) were analyzed using a Fluid Imaging Technologies FLOWCAM® PV-100 system. Samples for analysis were reconstituted to the desired concentration with filtered water (MILLI-Q®) in a sterile centrifuge tube. The samples were held in 2 mL Schott adaptiQ® (SCHOTT, Pharma) glass vials using an OmniFlex® (Daetwyler Holding Inc.) stopper and crimper. Three sets of samples were then examined. These included (i) a sample of the diluent used for reconstitution, (ii) an aliquot of the feed solution used for the particle formation process, i.e., a sample of the first aqueous liquid, and (iii) the reconstituted material.

[0227] Accelerated Storage: Unless otherwise specified, storage was carried out under accelerated conditions for selected samples by maintaining them at a high temperature (40°C) in an incubator or oven for a specified period. Samples were held in 2 mL or 4 mL WHEATON® glass vials and sealed with paraffin film.

[0228] Reverse Gas Chromatography (IGC): Powdered samples were analyzed using reverse gas chromatography. A stationary phase was prepared by packing a cylindrical column with 200-300 mg of powdered sample. After purging with an inert gas, a series of gas probes were injected onto the column. Determining the retention volume for each probe allowed for evaluation of the surface energy dispersibility and polarity components for each sample.

[0229] X-ray diffraction (XRD): The sample was packed into a 0.7 mm diameter glass capillary. The powder pattern was measured on a PANALYTICAL EMPYREAN® diffractometer equipped with an incident beam focusing mirror and an X'CELERATOR® detector. The pattern (1–50°²θ, 0.0167113° step, 4 sec / step, 1 / 4° divergent slit, 0.02 radian solar slit) was measured using Mo Ka irradiation. If electrostatic effects (which occurred after grinding in a mortar and pestle when evaluating freeze-dried controls) prevented the sample from being packed into the capillary, the powder pattern was measured from a flat plate specimen on a BRUKER® D2 Phase diffractometer equipped with a LYNXEYE® position-sensitive detector. The pattern was measured at 0.0202144° steps using Cu Kα irradiation at 5–100°²θ, counted at 1.0 sec / step. Standard instrument settings were used (30kV, 10mA, 0.6mm divergent slit, 2.5° solar slit, and 3mm scattering screen height).

[0230] Microfluidized Particle Sizing (MPS): Fluid imaging microscopy for particle size analysis was performed using FLOWCAM® PV-100. To investigate particle size and dispersibility, 5 mg of powder was dispersed in 10 mL of dry isopropanol via sonication. The continuous isopropanol phase prevented particle dissolution, i.e., prevented reconstitution. 0.3 mL was injected into the cell, and particle images were acquired using a flow rate of 0.15 mL / min. Particles with a roundness greater than 0.9 were reported in the analysis, and any double images were removed from the analysis to obtain the particle size distribution and dispersibility in the range of 1–100 μm.

[0231] Dynamic Vapor Adsorption (DVS): The powder was analyzed using dynamic vapor adsorption. Approximately 50 mg of the powdered sample was placed in the pan of the instrument's microbalance. The sample was kept isothermal at 22°C, and the sample mass was monitored throughout the measurement. After a 0% RH purge to remove surface water, the relative humidity (RH) in the sample chamber was gradient from 4% RH per hour to 90% RH. The sample was held at 90% RH for 1 hour, and then the RH was reduced to 0% in a stepwise manner. The sample was held at 0% RH for 1 hour, and then the measurement was terminated.

[0232] Dynamic Scanning Calorimetry (DSC): Powdered samples were analyzed using dynamic scanning calorimetry. 5–10 mg of powdered sample was packed into an aluminum crucible and airtightly sealed. The crucible was placed in the instrument, and the heat flow to the sample was monitored while the temperature was gradientned at a constant rate of 5°C / min from -80 to 200°C, and optionally from 20 to 180°C.

[0233] USP <790> :USP <790> According to the standard, samples of dissolved particles were visually observed against white and black backgrounds under illumination conditions exceeding 2000 lux. A matte-finish high-density polyethylene sheet was selected for the background to reduce glare. Illuminance at the observation point was confirmed using a lux meter (Dr. Meter, LX1330B). After mixing the samples, they were held against the background and visually observed for 5 seconds.

[0234] Aggregation volume: Aggregation volume can be measured by suspending particles in a desired liquid carrier at a suitable concentration. The suspension is then stirred, typically by shaking or mechanical means, to ensure uniform dispersion of particles in the fluid at the start of the measurement. Measurement begins immediately after stopping the stirring. The mixture is allowed to stand, and the degree of solid sedimentation or settling is observed and recorded over time.

[0235] Example 1 General protocol for preparation of protein particles containing hyaluronidase: An aqueous protein solution was prepared to a final concentration of 50–180 mg / mL. An aqueous solution of excipients and hyaluronidase (stock concentration of hyaluronidase at 2000 U / mL, 0.000051–8.81% w / w; for particle loads of 375–750 mg / mL) was added to the obtained solution and filtered through a small 0.22 μm filter to remove any exogenous solids from the obtained "feed" aqueous solution. The protein feed solution and dehydrating solvent (e.g., n-butyl acetate) were mixed at different flow rates (1 mL / min–10 L / min) to produce aqueous droplets, which were then dehydrated (time dependent on the volume of dehydrating solvent, rpm of the mixture, and flow rate) to form protein particles suspended in the mixture. The protein particles were separated from the liquid mixture (e.g., by filtration, centrifugation, and decantation). The liquid mixture (aqueous liquid and dehydrating solvent) was removed. Residual solvents (e.g., aqueous liquid and dehydrating solvent) in the protein particles were removed (e.g., vacuum drying, gas drying, gas sparging, extraction solvent, etc.) and the water content in the particles was adjusted (e.g., humidifying gas, extraction solvent, etc.). Optionally, a portion of the protein particles were dissolved in DI water and incubated to fully redissolve for characterization. The solutions were analyzed to measure protein quality (SvP analysis using FLOWCAM®, and soluble coagulation (e.g., SEC), fragmentation (e.g., SEC), charge variants (e.g., changes in ion exchange chromatography (e.g., strong cation exchange chromatography (SCEX) analysis)), as well as protein concentration. Protein concentrations in the range of 200 mg / mL to 800 mg / mL were processed according to a general protocol (at least 60–93% (w / w) particle protein content). Particles with a maximum protein content of 85% showed improved stability compared to the initial aqueous supply solution. Generally, the amount of hyaluronidase in the suspension is approximately 20 U / mg of protein.

[0236] Preparation of Ab particles in the target size and dispersibility range: Rituximab, trastuzumab, and human or bovine IgG particles were formed under a general protocol, and 10 mg of particles were provided for analysis. Average particle sizes, D10, D50, and D90 were reported based on count-weighted particle size analysis (FLOWCAM® or optical diffraction). The average particle sizes, D10, D50, and D90 of the particles were determined to be 5–50 μm and D90 < 60 μm. The particles had the following particle size statistics: average diameter = 5–50 μm. SEM of particles for each batch was obtained. Images were acquired at magnifications up to 4000x, as shown in Figure 1. Image processing and statistics were performed using ImageJ analysis, count-weighted, to provide protein particles with desirable physical characteristics (e.g., roundness, internal void space, protein by weight, stability, and quality) used for suspension studies. Figure 2 is a FIB-SEM image showing that the microparticles containing trastuzumab and hyaluronidase are smooth, spherical, and free of any voids. The roundness of the particles ranged from 0.99 to 1.00.

[0237] Figure 3 shows particle sizes (D10, D50, and D90) and particle spans containing trastuzumab, rituximab, mixed antibody, and BSA suspended in PGD or EO. The non-precipitating suspension is a particle suspension with a flocculant, as shown in Figure 3. In some embodiments, the flocculant may be PS80. The particle sizes and spans are within the expected range so that the suspension can flow through the syringe without clogging. The particle span is calculated using the following formula:

number

[0238] Determination of coagulation, fragmentation, and charge variant changes in hyaluronidase-containing particles by SEC: Dry powder samples containing hyaluronidase were dissolved in ultrapure water to 5% (w / v) and shaken at 60 RPM. After determining the protein concentrations in triplicates, the samples were diluted to 1 mg / mL in PBS and syringe filtered into HPLC vials. The samples were resuspended in the carrier liquid, and each sample was immediately centrifuged at 500 RPM. The resulting carrier liquid supernatant was aspirated (and discarded) without disturbing the settled particles. After adding water to the particles and mixing, the samples were shaken at 60 RPM. After determining the initial protein concentrations (from the bottom or aqueous layer), each sample was diluted in PBS and the protein concentrations were measured in triplicates (from the bottom or aqueous layer). Then, each sample was diluted in PBS and syringe filtered into HPLC vials, discarding the needle before filtration (to prevent transfer of the carrier liquid). All samples were analyzed using an AGILENT® 1260 Infinity II bioinert LC system and a TSKgel SuperSW HTP column (4 μm, 4.6 mm ID × 150 mm L) equilibrated with 200 mM arginine-HCl and 100 mM sodium phosphate, pH 6.5. The autosampler and column compartment were maintained at 4°C and 20°C, respectively, and UV absorbance was monitored at 280 nm. The run time was 10 minutes. Area percentages of integrated high molecular weight (coagulated), monomer, and low molecular weight peaks are reported.

[0239] Determination of coagulation, fragmentation, and charge variant changes in hyaluronidase-containing particles by SCEX: Dry powder samples containing hyaluronidase were dissolved in ultrapure water to 5% (w / v) and shaken at 60 RPM. After determining the protein concentrations in triplicates, the samples were diluted with ultrapure water and syringe-filtered into HPLC vials. The samples were resuspended in the carrier liquid, and each sample was immediately centrifuged at 500 RPM. The resulting carrier liquid supernatant was aspirated (and discarded) without disturbing the settled particles. After adding water to the particles and mixing, the samples were shaken at 60 RPM. After determining the initial protein concentration (from the bottom or aqueous layer), each sample was diluted with ultrapure water and the protein concentration was measured in triplicates (from the bottom or aqueous layer). Then, each sample was diluted with ultrapure water and syringe-filtered into HPLC vials, discarding the needle before filtration (to prevent transfer of the carrier liquid). All samples were analyzed using an AGILENT® 1260 Infinity II bioinert LC system and a THERMO SCIENTIFIC MABPAC® SCX-10 RS column (5 μm, 2.1 mm ID × 150 mm L) equilibrated with 100% mobile phase A (THERMO SCIENTIFIC® 1X CX-1 pH gradient buffer A). The gradient was 0–20% mobile phase B (THERMO SCIENTIFIC® 1X CX-1 pH gradient buffer B). The autosampler and column compartment were maintained at 4°C and 30°C, respectively, and UV absorbance was monitored at 280 nm. The area percentages of integrated acidic, neutral (major), and basic peaks were reported.

[0240] Sub-Visible Particle Analysis: Sub-visible particulate matter present in dissolved particles was investigated. Sub-visible particle analysis was performed after particle dissolution. This data was compared with other competitive particle formation techniques. SvP analysis was performed using a particle analyzer, where the particle count was corrected for background signals in the control sample. The sub-visible particle count measured in the sample was lower than that of any other finished technique, including standard lyophilization.

[0241] Turbidity: Aliquots of the reconstituted particle solution were transferred to 1-cm cuvettes. Absorbance at 405 nm was recorded using a NANODROP® One UV-VIS spectrophotometer (THERMO SCIENTIFIC®). The results showed similar turbidity compared to the initial aqueous supply solution and particles.

[0242] Determination of hyaluronidase activity in particles: A hyaluronidase activity ELISA assay was performed for the detection and quantification of hyaluronidase activity ((K6000 kit) Echelon Biosciences Inc., Salt Lake City, Utah). The hyaluronidase reaction was carried out in a 96-well microtiter plate pre-coated with hyaluronane substrate and incubated for at least 30 minutes. Hyaluronidase activity in particles and aqueous supply solution was determined by comparing the hyaluronane substrate level after the reaction with a standard curve of pre-coated hyaluronane substrate volume. Supply and dissolution suspensions containing the hyaluronidase enzyme were diluted to approximately 1 U / mL of enzyme and tested on the kit. Successful removal of bound hyaluronic acid was reported as the percentage of digestion (digestion %), indicating the active enzyme. A 1 U / mL positive control sample showed 100% removal of hyaluronic acid and 100% digestion. The aqueous supply solution was tested at a concentration of 30 mg / mL, and the suspension particles were also tested at a concentration of 30 mg / mL. The dry powder sample was dissolved in ultrapure water to a concentration of 30 mg / mL. Hyaluronidase in the aqueous supply solution (3.90) and particle solution (3.69) showed retained enzyme activity compared to the control sample, as shown in Figure 4.

[0243] Figure 5 shows the digestion rate (i.e., the activity of hyaluronidase for digesting hyaluronic acid) for both the aqueous formulation (supplied) containing trastuzumab and the suspension. A higher digestion rate indicates better activity. Compared with the aqueous formulation (supplied), the enzyme activity showed increased retention in the microparticle suspension after 6 months of storage. The stability of the hyaluronidase enzyme is retained in the microparticles over 6 months. The stability of the enzyme in the suspension is maintained at 97% after accelerated storage in the suspension at 40°C for 6 months, compared to 91% for the aqueous formulation.

[0244] According to Figure 6, hyaluronidase enzyme activity is maintained in formulations containing two therapeutic monoclonal antibodies (trastuzumab and rituximab) compared to the aqueous formulation (supplied). The enzyme activity of the samples is retained for 3 months at 4°C and 40°C, demonstrating stability over 3 months.

[0245] Smaller proteins such as bovine serum albumin (BSA) can be formulated into microparticle suspensions (Figure 7). Enzyme activity was largely retained by the treatment. After storage at 4°C and 40°C for 3 months, there is a slight decrease in activity in both the suspension and the aqueous formulation.

[0246] Example 2 General protocol for protein particles having a hyaluronidase composition: Rituximab particles, trastuzumab particles, or IgG (human or bovine) particles containing excipients (e.g., histidine, arginine.HCl, PS80, PS20, trehalose, NaCl, sucrose, methionine, proline, sodium phosphate) and hyaluronidase were made into a suspension at a protein concentration of 200 to 700 mg / mL with a hyaluronidase concentration of up to 1800 or 2000 units / mL (less than 0.01%) in an organic carrier liquid or a mixture of at least two organic carrier liquids (e.g., ethyl oleate, sesame oil, medium-chain triglycerides (MCT), propylene glycol diesters (PGD) of saturated plant fatty acids C8 and C10, triacetin, caprylic acid triglycerides). This was achieved by adding the protein particles comprising hyaluronidase to the organic carrier liquid and generating a diluted suspension using a mixer (e.g., a rotor-stator or the like). The diluted suspension was sieved through a 60 μm filter, and the suspension was adjusted to the desired protein concentration. To this suspension, a flocculant was added to reach a total concentration of flocculant of up to 10 mg / mL at a protein concentration of 200 to 800 mg / mL. In general, the amount of hyaluronidase in the suspension is about 20 U / mg per mg of protein. This suspension was initially mixed using 1 minute of vortexing and / or mechanical mixing to create the desired aggregate volume that remains stable for at least one month.

[0247] HIgG particle composition: HIgG particles containing hyaluronidase (77% protein: 2% histidine: 20% arginine: 1% PS80 (or PS20): 0.006% hyaluronidase) were prepared as a suspension at a concentration of 300 mg / mL in ethyl oleate. This was achieved by adding 1.8 g of particles to approximately 120 mL of ethyl oleate and preparing a diluted suspension using a rotor-stator mixer. The diluted suspension was sieved through a 60 μm filter and adjusted to a concentration of 300 mg / mL of HIgG by centrifugation. Optionally, a coagulant (polysorbate 80) was pipetteed into aliquots of this suspension to reach a final total coagulant concentration of up to 10 mg / mL at an HIgG concentration of 300 mg / mL. This suspension was vortexed and then mechanically mixed for 1 minute to produce a stable suspension (coagulation volume) that maintained consistency for at least 1 month compared to a particle suspension without a coagulant. HIgG and BIgG were used interchangeably. Protein characterization was achieved according to Example 1, showing improved stability compared to the initial aqueous supply solution.

[0248] Example 3 Protein and hyaluronidase particle compositions having high concentration, stable aggregate volume, and low injection force: The units "mPa·s" and "cP" are used interchangeably in the broadest sense herein.

[0249] Rheology: Suspensions of rituximab, trastuzumab, bovine IgG, or human IgG and hyaluronidase particles were prepared with the following characteristics: protein content >250 mg / mL and apparent viscosity <20 cP (mPa·s). Viscosity was measured using a parallel plate rheometer or other methods known in the art. Suspensions were prepared at various concentrations in a carrier liquid. Viscosity of approximately 20 cP (mPa·s) was measured at a concentration >300 mg / mL. Higher concentrations can be achieved if carriers with lower viscosity are considered.

[0250] Rheology (Days 0, 7, and 30 at 40°C): The viscosity of the particle suspensions (rituximab, trastuzumab, bovine IgG, or human IgG and hyaluronidase) was tracked over time to ensure that it did not fluctuate by more than 5% over accelerated storage of 7 and 30 days. At each time point, the suspensions had a viscosity <20 cP (mPa·s) with a protein content >250 mg / mL. Concentration requirements were met based on the preparation of a 280 mg / mL protein suspension. Samples on day 0 were measured immediately. Samples on day 7 and 30 were aged according to the aging procedure outlined above. 1000 seconds -1 Measurements were taken at the shear rate. The viscosity of the suspension was measured on days 0, 7, and 30. Generally, there was no change in the viscosity of the suspension at day 30.

[0251] Karl Fischer titration: The water content of solid particles (rituximab, trastuzumab, bovine IgG, or human IgG and hyaluronidase) was determined, and the composition of the particle components was defined using Karl Fischer titration. The water content of the particles was measured and recorded, and it was determined to be between 1 and 6% by weight for each of rituximab, trastuzumab, bovine IgG, or human IgG.

[0252] Viscosity determination: A 25 mm 3-degree cone (CP25-3, Anton Paar) was loaded onto a rheometer (Anton Paar Modular Compact Rheometer (MCR) Series 92). The plate was warmed to 25°C. The suspension containing the flocculant was vortexed until visually homogeneous. 250 μL was pipetteed onto the base plate, and the sample was measured at a shear rate of 950 1 / s.

[0253] Determination of protein content: A particle suspension containing a flocculant was dispersed by vortexing, diluted to 2.5% (v / v) in ultrapure water, and shaken at 60 RPM for 30 minutes. The corresponding dry powder sample was dissolved in ultrapure water to 5% (w / v) and shaken at 60 RPM for 30 minutes. After determining the triple protein concentrations (from the bottom or aqueous layer of the suspension), the mass percentage of protein in the particles was calculated by dividing the product of the averaged redissolved protein particle concentration and redissolved volume by the mass of the fine particles. Then, the theoretical mass of particles in the suspension was calculated by dividing the averaged concentration of the suspension (corrected by the aqueous / formulation carrier liquid dilution factor) by this mass percentage of protein in the particles. The results correspond to the protein content shown in Examples 1 and 2.

[0254] Dissolution: Dissolution rates were recorded for each suspension on days 0, 7, and 30. Dissolution was recorded at various time points, and dissolution was confirmed to occur within approximately 20 minutes for particles and approximately 60–80 minutes for suspensions. Particles and suspensions were dissolved in PBS to a final concentration of 10 mg / mL and shaken on a rocker. Dissolution of particles on days 0, 7, and 30 was also stored at 40°C.

[0255] USP <790> The presence of visible particles was determined in the sample in which the particles were dissolved. USP <790> We used [a specific method] to determine whether particles in the "visible" range (>100 μm) were present. The presence of visible particles was assessed using observations of dissolved particles. Observations were brief (5 seconds) with white and black backgrounds under appropriate lighting. Several potentially small particles were observed, but were difficult to see with the naked eye and could be classified as sub-visible particles. Given that these particles may be considered sub-visible, we investigated further using other methods as described below.

[0256] Sub-Visible Particle Analysis: Sub-visible particulate matter present in dissolved particles was investigated. Sub-visible particle analysis was performed after particle dissolution. This data was compared with other competitive particle formation techniques. SvP analysis was performed using a particle analyzer, where particle counts were adjusted for background signals in the control sample. The sub-visible particle counts measured in the sample were lower than those of any other finished technique, including standard lyophilization.

[0257] Aggregation volume: Protein particles were added to 3 mL of ethyl oleate, followed by the addition of 0.1% to 0.01% by weight of a flocculant (lecithin or PS80) to form a protein concentration of at least 400 mg / mL with 2000 U / mL of hyaluronidase. The suspensions were initially mixed, the aggregation volume was calculated, and compared with the suspension without the flocculant. Generally, in most cases, no reduction in aggregation volume was observed, and the suspension remained stable at 25°C for at least 3 months.

[0258] Injectability: The force required to inject a suspension containing a flocculant through a needle was measured, and it was verified that the injection force remained the same 24 hours after injection without resuspension. As obtained above, a suspension of protein particles with a concentration of at least 400 mg / mL of protein in EO and 0.1 wt% of PS80 was filled into a 5 mL syringe with an 18 G needle (3 inch). The injection force was less than 2.0 N and remained the same after 24 hours of storage. For reference, the force required to actuate an empty syringe with a syringe needle was compared using a 15 mPa·s standard, and the results showed that the injection force was generally below 2.0 N. These studies demonstrate that the force required to inject the composition remained stable for at least one month.

[0259] Example 4 Particle and suspension quality studies: Design: A suspension of particles containing a flocculant was obtained using the same procedure as in Examples 1 and 2. The stability of the protein and hyaluronidase particle suspensions containing the flocculant was evaluated over periods of up to 3 months at 5, 25, and 40°C to determine the stability of the particle suspension composition compared to the protein liquid drug substance (LDS), for example, compared to the initial aqueous supply solution.

[0260] Composition: Using the same procedure as in Examples 1 and 2, a suspension of particles containing a formulated flocculant (600 mg / mL protein, 2000 U / mL and 0.1 mg / mL PS80; 74% protein: 2% histidine: 24% arginine: 1% PS80) was obtained, and then suspended in ethyl oleate.

[0261] Using the same procedure as in Examples 1 and 2, a suspension of particles containing a formulated flocculant (600 mg / mL protein, 20 U / mg and 0.1 mg / mL PS80; 86% protein: 2% histidine: 12% arginine: 1% PS80) was obtained, and then suspended in either PGD, ethyl oleate, MCT, or a mixture of ethyl oleate and MCT. In some embodiments, the mixture may be in a 1:1 ratio.

[0262] Buffer exchange by tangential flow filtration (TFF): Protein and hyaluronidase feed solutions were prepared by diafiltration into the required buffer cocktail. Feed preparation was performed using a KrosFlow KR2i TFF system (Repligen) equipped with a hollow fiber filter module (MiniKros Sampler). Several diafiltration volume exchanges were performed with the appropriate buffer for each formulation.

[0263] FIB-SEM imaging: Particles were imaged using a scanning electron microscope (HITACHI™, TM-1000). The particle sample was placed on an adhesive stage for analysis. Images were captured at various magnifications using an acceleration voltage of 15 kV.

[0264] Buffer exchange by tangential flow filtration (TFF): Protein and hyaluronidase feed solutions were prepared by diafiltration into the required buffer cocktail. Feed preparation was performed using a KrosFlow KR2i TFF system (Repligen) equipped with a hollow fiber filter module (MiniKros Sampler). Several diafiltration volume exchanges were performed with the appropriate buffer for each formulation.

[0265] SEM imaging: Particles were imaged using a scanning electron microscope (HITACHI™, TM-1000). The particle sample was placed on an adhesive stage for analysis. Images were captured at various magnifications using an acceleration voltage of 15 kV.

[0266] Karl Fischer coulometric measurement: Testing for water content was performed by Karl Fischer analysis using a MetroOhm (899 Coulometer) equipped with an 860 KF THERMOPREP™ oven. The particles were heated to 165°C in the oven, and the released water was determined coulometrically.

[0267] Particle dissolution: Water was added to the dry particle sample to produce the final protein concentration. The sample was placed in a nutating mixer at 60 RPM for a set period of time. The final dissolved concentration was recorded by taking an aliquot from the sample and measuring the absorbance at 280 nm (using the extinction coefficient E1% = 1.69 L·g⁻¹·cm⁻¹).

[0268] Turbidity: Aliquots of the reconstituted particle solution were transferred to 1-cm cuvettes. Absorbance at 405 nm was recorded using a NANODROP® One UV-VIS spectrophotometer (THERMO SCIENTIFIC®). The results generally showed improved turbidity compared to the initial aqueous supply solution and particles.

[0269] Size exclusion chromatography (SEC) measurement: Sample injection was performed on a Tosoh TSKgel SuperSW mAb HTP (4.6 mm ID × 15 cm L) column at a flow rate of approximately 0.35 mL / min for 10 minutes using a mobile phase consisting of 100 mM sodium dihydrogen phosphate and 200 mM L-arginine monohydrochloride, pH 6.5. Peaks were manually inspected to ensure accurate identification, and analysis was performed by automated integration using parameters known in the art.

[0270] Intensive cation exchange chromatography (SCEX): Samples dissolved in water were injected at a flow rate of 0.4 mL / min using a gradient method, starting with 100% mobile phase pH gradient buffer A into 100% mobile phase pH gradient buffer B, for a total run time of 40 minutes on a MAbPac® SCX-10 RS analytical column, 2.1 mm ID × 15 cm L, 5 μm column, followed by washing and re-equilibriumization. Peaks were manually examined to ensure accurate identification, and analysis was performed by automated integration using parameters known in the art.

[0271] Hydrophobic Interaction Chromatography (HIC): Samples dissolved in a diluent consisting of 750 mM ammonium sulfate, 50 mM sodium dihydrogen phosphate dihydrate, pH 6.0 (1 mg / mL) were injected onto a MAbPac® HIC-20 HPLC column, 5 μm, 4.6 mm ID × 25 cm L column for a total run time of 60 minutes. The process was performed using a gradient method at a flow rate of 1 mL / min, starting with 100% mobile phase B consisting of 50 mM sodium dihydrogen phosphate dihydrate, pH 6.0, followed by 50% mobile phase A consisting of 2 M ammonium sulfate, 50 mM sodium dihydrogen phosphate dihydrate, pH 6.0, and then washing and re-equilibriumization with 50% mobile phase A. Peaks were manually inspected to ensure accurate identification, and analysis was performed by automated integration using parameters known in the art.

[0272] Particle sizing (laser diffraction): Particle size analysis was performed via laser diffraction using a Horiba LA-960S. Dry particles were suspended in isopropyl alcohol at a concentration of approximately 0.1 mg / mL. The particle suspension was sonicated in the particle measuring instrument to ensure uniformity, and then circulated and stirred using a Horiba particle size analyzer. Particle size analysis was performed using isopropyl alcohol as the mobile phase, and the volume-averaged particle size distribution was calculated.

[0273] Microfluid Imaging: Fluid imaging microscopy (FLOWCAM®, Fluid Imaging Technologies) was performed to quantify sub-visible particles in protein LDS and particle formulations. Particles were first redissolved using the particle lysis method described above and diluted to 1 mg / mL with ultrapure water. For analysis, aqueous samples were introduced at a flow rate of 0.15 mL / min. The resulting particle counts were recorded and reported per mg of protein.

[0274] Results: The stability of dry particles and particle suspensions with flocculants was evaluated in parallel with that of protein liquid drug substances (LDS). Stability was tracked at 5°C, 25°C, and 40°C, and data were collected at 7, 14 days, 1, 3, 6, 9, and 12 months. Particle stability, protein and hyaluronidase stability of LDS, and suspension stability were measured at each time and temperature. Analysis of particles and particle suspensions confirmed that the quality of protein and hyaluronidase remained constant, as measured by monomer profile (SEC), maintenance of charge variant profile (CEX), isoform / presumed oxidation (HIC), and colloidal stability (turbidity and sub-visible particles). In each case, the protein and hyaluronidase supply solutions were measured against particles and particle suspensions with flocculants. Particles stored as dry powder and particle suspensions at 5°C, 25°C, and 40°C exhibited a smooth, spherical morphology. At these temperatures, no changes in particle morphology or particle size distribution were observed over a period of at least 3 months. The moisture content of the particles remained constant for all storage temperatures and durations. Analysis of protein coagulations was performed by SEC at 5°C, 25°C, and 40°C for up to 12 months. For standards, the effect of excipient selection (histidine, arginine HCl, and PS80) on the stability of protein particles (without flocculants) for Δ(Mon+Dim)% and Δ(Dim+Agg)% was evaluated, and compared with LDS samples, coagulation was assessed over time after storage at 40°C for at least 3 months. In general, LDS samples stored at 40°C for at least 3 months showed higher protein coagulation compared to suspensions with protein particles and flocculant samples stored at the same temperature (40°C).

[0275] The lower rate of coagulation in particle suspensions with flocculants indicated improved stability compared to protein LDS. The protein charge variant profile was measured by SCEX at 5°C, 25°C, and 40°C for up to 90 days. No recognizable changes were observed for particles and particle suspensions with flocculants, which showed improved stability at 40°C compared to protein LDS. Potential oxidation of the protein was measured using HIC. The estimated overall degree of oxidation of the protein was measured by HIC at 5°C, 25°C, and 40°C for up to 90 days. No recognizable changes were observed, indicating that particles and particle suspensions with flocculants showed improved stability with respect to oxidative stress at 25°C and 40°C compared to protein LDS at the same temperature and time. To further explore the protein quality of redissolved particles in aqueous media, visible and sub-visible particles were analyzed after storage at various temperatures. Upon dissolution, the protein solution essentially contained no visible or sub-visible particles after storage at 40°C for up to 90 days. For particle suspensions containing a flocculant, protein concentration, hyaluronidase activity, viscosity, and injection force did not change significantly at all storage times and temperatures.

[0276] Figure 8 shows that the stability of the BSA protein is maintained in microparticles during processing. After storage at 4°C and 40°C for 3 months, the BSA protein quality attributes (i.e., HMW%, charge variants (basic% and acidic%), and sub-visible particles (>2um)) are similar to those of the aqueous formulation (supply). Formulations with and without hyaluronidase have similar protein quality, indicating that the addition of the enzyme does not affect the quality of the therapeutic protein. Samples with and without hyaluronidase had similar stability profiles for BSA formulated as a suspension in PGD (Figure 8).

[0277] Figure 9 shows that trastuzumab samples with and without hyaluronidase exhibit similar levels of coagulation (HMW%) and fragmentation (%LMW). When comparing aqueous formulations (supply) with suspension formulations (suspension-PGD) and suspensions with coagulant formulations (non-precipitating suspension-PGD, non-precipitating suspension-EO), less than 10% coagulation and virtually no fragmentation of trastuzumab were observed after treatment.

[0278] Figure 10 shows that trastuzumab samples with and without hyaluronidase exhibit similar trastuzumab charge variant profiles. No substantial changes were observed after treatment when comparing aqueous formulations (supply), suspension formulations (suspension-PGD, without flocculant), and suspension formulations with flocculants (non-precipitating suspension-PGD, non-precipitating suspension-EO).

[0279] Figure 11 shows the profiles of sub-visible particles derived from trastuzumab samples with and without hyaluronidase. When comparing the aqueous formulation (supply) with a suspension without a flocculant (suspension-PGD) and a suspension with a flocculant formulation (non-settling suspension-PGD), an increase in SVPs with sizes greater than 10 μm and greater than 25 μm was observed.

[0280] Figure 12 shows the percentage of monomers and neutral species obtained from trastuzumab samples with and without hyaluronidase before and after storage at 4°C and 40°C for 3 and 6 months, respectively. During 6 months of storage, the suspension exhibited better stability than the aqueous formulation. Accelerated storage (40°C) showed that the aqueous formulation reduced trastuzumab monomer to 86.7% and neutral species to 21%. For hyaluronidase, the suspension formulations with and without the PS80 flocculant contained 91.6% and 91.3% trastuzumab monomer, respectively, while the neutral species accounted for 39.2% and 37.2%.

[0281] Cake height measurements of trastuzumab suspensions compared to non-sedimenting suspensions show that adding the PS80 surfactant as a flocculant to the carrier liquid prevents particle sedimentation during storage at room temperature for 3 months (Figure 13). The suspension without hyaluronidase showed a cake height of 85.6% after 3 months of storage at 25°C, while the suspension containing hyaluronidase showed a cake height of 86.6%. The suspension with the flocculant showed a cake height of 100%.

[0282] Cake height measurements of trastuzumab and rituximab (mixed antibody) suspensions compared to non-precipitating suspensions show that adding PS80 surfactant as a flocculant to the carrier liquid prevents particle sedimentation over 48 hours at room temperature (Figure 14). The suspensions showed a cake height of 88.2% after storage at 25°C for 48 hours.

[0283] Figure 15 shows the cake height measurements of a trastuzumab particle suspension (containing hyaluronidase) compared to a non-precipitating suspension (i.e., a suspension with PEG400 flocculant) after storage at 25°C for 3 days. The cake height of the suspension without flocculant was 73% compared to 100% of the suspension containing PEG400 flocculant. PEG400 is present in the carrier liquid and not in the aqueous solvent (e.g., not used as a crowding agent).

[0284] Example 5a Pharmacokinetics (PK): Subcutaneous administration of Ab and hyaluronidase microparticle suspensions shows higher bioavailability than administration of Ab microparticle suspension alone. Particle and droplet generation (Silverson / 20mm) studies: dispersed phase:continuous phase (nBA) ratio 1 / 200, with mixing of at least 16 mL:3.2 L, and a mixer speed of (4000 rpm) / 50 sec. Solid-liquid separation process by centrifugation and subsequent drying (air 10-75 RH) provided smooth, spherical particles with a particle size distribution (D10 / D50 / D90) of 9.39 / 16.79 / 29.15 μm. SVP > 2 μm (610975 particles) (3.5E5), normalized to 1 mg protein per Aura, Pro-A, 16.00% oxidation, SCEX (% acidic / neutral / basic) 7.7 / 82.7 / 9.6, SEC showed 4.7% high molecular weight aggregates, 94.6% monomer data, and 0.72% low molecular weight data, indicating that the particles remained stable for at least 3 months.

[0285] Particle formulation: HIgG particles containing hyaluronidase (77% protein: 2% histidine: 20% arginine: 1% PS80 (or PS20): 0.006% hyaluronidase) were prepared as a suspension at a concentration of 300 mg / mL in ethyl oleate. This was achieved by adding 1.8 g of particles to approximately 120 mL of ethyl oleate and producing a diluted suspension using a rotor-stator mixer. Generally, the amount of hyaluronidase in both suspensions is approximately 20 U / mg per 1 mg of protein.

[0286] Composition: The scope of this study includes two cohorts having the following compositions. 1) SC Ab protein and hyaluronidase microparticle suspension: 80 mg / mL HIgG, 1600 U / mL hyaluronidase, 2.5 mg / mL histidine, 20 mg / mL arginine, and 0.5 mg / mL polysorbate 80 were formulated into a particle composition (77% protein, 0.006% hyaluronidase), and then suspended in ethyl oleate (non-aqueous carrier vehicle) at a protein concentration of 300 mg / mL. 2) SC Ab protein microparticle suspension: 80 mg / mL HIgG, 2.5 mg / mL histidine, 20 mg / mL arginine, and 0.5 mg / mL polysorbate 80 were formulated into a particle composition (77% protein, 0.006% hyaluronidase), and then suspended in ethyl oleate (non-aqueous carrier vehicle) at a protein concentration of 300 mg / mL.

[0287] Animals: Male Wistars rats (Harlan Laboratories) were housed in the animal facility at Tufts University Comparative Medicine Services (Tufts CMS). All use of the described animals was carried out in accordance with the U.S. National Research Council's "Guide for the care and use of laboratory animals" and in accordance with detailed written protocols approved by the Animal Care Committee (IACUC).

[0288] Dosage, study design, and analysis: All particulate composition injections were achieved using 100 μL Hamilton glass syringes with 27 gauge UTW needles. All aqueous and non-aqueous vehicle injections were achieved using 300 μL syringes with staked-in 30 gauge UTW needles. Male Wistars rats were used for this study. The following study designs were used: 1) Ab protein and hyaluronidase microparticle suspension; dose = 20 mg / kg protein, 2000 U / mL hyaluronidase; volume = 13 μL; n = 5. 2) Ab protein microparticle suspension; dose = 20 mg / kg protein; volume = 13 μL; n = 5. Plasma samples were collected at 0h, 3h, 6h, 9h, 1d, 2d, 3d, 5d, 6d, 7d, 9d, 12d, 14d, 16d, 19d, 21d, and 23d. At these time points, the detection limits of the assay used were below. Blood from each rat was collected from tail vein bleeding into EDTA capillary tubes (purple) at all data points. Plasma obtained from the blood was diluted 10,000-fold for analysis using ELISA. Plasma samples were evaluated using an anti-human IgG ELISA assay (Ray Biotech: RayBio® Human IgG ELISA Kit) performed by a third-party contract laboratory. SC injection of HIgG with hyaluronidase microparticle suspension in ethyl oleate (EO) of the test article: AUC 25.7 (h* mg / mL), Cmax 125.4 (μg / mL), Tmax 72.0 (h). The HIgG microparticle suspension SC injection in ethyl oleate (EO) of the test material showed an AUC of 20.6 (h*mg / mL), a Cmax of 99.0 (μg / mL), and a Tmax of 72.0 (h). As shown in Figure 16, HIgG with hyaluronidase microparticle suspension exhibits higher bioavailability than HIgG microparticle suspension injection.

[0289] Toxicity and local tolerability studies and analyses: Injection sites were evaluated for edema (swelling) and erythema (redness) before and for up to 3 days after injection. Draize scoring was used to score sites based on the severity of the response (maximum 0-4). No abnormal findings were observed on day 0 (before injection), day 1 (after injection), and day 7 (after injection) in any of the five animals in the composition groups (100 mg / kg, 500 mg / mL).

[0290] Erythema and crusting: No erythema - 0; Very mild erythema (barely perceptible) - 1; Well-defined erythema - 2; Moderate to severe erythema - 3; Severe erythema (beef redness) to crusting that interferes with erythema grading - 4.

[0291] Edema formation: No edema - 0; Very mild edema (barely perceptible) - 1; Mild edema (border of area clearly defined by a distinct elevation) - 2; Moderate edema (elevation of approximately 1 mm) - 3; Severe edema (elevation greater than 1 mm and expansion beyond the exposed area) - 4.

[0292] Draize scoring: The presence or absence of findings was recorded for each individual animal on day 0 (pre-injection), day 1 (post-injection), and day 7 (post-injection). All five animals in the group had zero edema and erythema scores over the three days post-injection, indicating that the composition sample was well-tolerated without toxicity.

[0293] Detailed clinical observations: Animals were removed from their cages and observed. These included, but were not limited to, evaluations of the effects on the skin, fur, eyes, ears, nose, mouth, chest, abdomen, genitals, limbs and feet, respiratory and circulatory systems, autonomic nervous system effects such as salivation, and nervous system effects including tremors, convulsions, responsiveness to handling, and abnormal behavior. No abnormal findings were observed in any of the five animals in the composition groups (100 mg / kg, 500 mg / mL) on day 0 (before injection), day 1 (after injection), and day 7 (after injection), indicating that the composition samples were well tolerated without observable toxicity.

[0294] Example 5b Pharmacokinetics (PK): Subcutaneous administration of Ab and hyaluronidase microparticle suspensions shows higher bioavailability than administration of Ab microparticle suspension alone. Particle and droplet generation studies: Dispersed phase:continuous phase (nBA) ratio 1 / 200, at least 16 mL:3.2 L. Solid-liquid separation process by centrifugation and subsequent drying by solvent extraction provided smooth, spherical particles with an average D50 of 20 μm. SVP > 2 μm (3478 particles), SCEX (acidic / neutral / basic%) 7 / 51.6 / 41.4, normalized to 1 mg protein by Aura, SEC shows 1.29% high molecular weight aggregates, 98.6% monomer data, and 0.076 low molecular weight data.

[0295] Particle formulation: Trastuzumab particles containing hyaluronidase (86% protein: 2% histidine: 12% arginine: 1% PS80 (or PS20): 20 U / mg of hyaluronidase) were prepared in a 500 mg / mL suspension in PGD. Generally, the amount of hyaluronidase in a suspension is approximately 20 U / mg per 1 mg of protein.

[0296] Composition: The scope of this study includes two cohorts having the following compositions. 1) SC Ab protein and hyaluronidase microparticle suspension: 100 mg / mL trastuzumab, 1600 U / mL hyaluronidase, 2.5 mg / mL histidine, 12 mg / mL arginine, and 0.1 mg / mL polysorbate 80 were formulated into a particle composition (86% protein, 0.006% hyaluronidase), which was then suspended in PGD (non-aqueous carrier vehicle) at a protein concentration of 500 mg / mL. 2) SC Ab protein microparticle suspension: 100 mg / mL trastuzumab, 2.5 mg / mL histidine, 20 mg / mL arginine, and 0.1 mg / mL polysorbate 80 were formulated into a particle composition (86% protein, 0.006% hyaluronidase), which was then suspended in PGD (non-aqueous carrier vehicle) at a protein concentration of 500 mg / mL.

[0297] Animals: Gottingen miniature pigs were also used to test for microparticle and hyaluronidase activity. Animal studies were conducted by staff at the Charles River Laboratory in accordance with detailed written protocols approved by the Animal Research Committee (IACUC).

[0298] Dosage, study design, and analysis: All particulate composition injections were performed using a 1 mL Airtite syringe with a 25 gauge UTW needle. Female Gottingen miniature pigs were used in this study. The following study designs were used: 1) Ab protein and hyaluronidase microparticle suspension; dose = 14.4 mg / kg protein, 20 U / mg hyaluronidase; 2) Ab protein microparticle suspension; dose = 14.4 mg / kg protein. Plasma samples were collected at 0h, 6h, 1d, 2d, 3d, 5d, 7d, 9d, and 13d. Blood from each animal was collected from tail vein bleeding into EDTA capillary tubes (purple) at all data points. Plasma obtained from the blood was diluted 10,000-fold for analysis using ELISA. Plasma samples were evaluated using an anti-human IgG ELISA assay (Ray Biotech: RayBio® Human IgG ELISA Kit) performed by a third-party contract laboratory. As shown in Figure 17, the addition of hyaluronidase to the microparticles increased the bioavailability of the suspension in Gottingen miniature pigs. Figure 17 shows that the sample with hyaluronidase (blue trace) increased the plasma concentration of trastuzumab compared to the formulation without hyaluronidase (red trace). The increases in AUC and cMax (pharmacokinetic parameters) for the aqueous formulation after hyaluronidase addition were 14.8% and 38.4%, respectively. The increases in AUC and cMax (pharmacokinetic parameters) for the suspension formulation after hyaluronidase addition were 47% and 71.3%, respectively.

[0299] Toxicity and local tolerability studies and analyses: Injection sites were evaluated for edema (swelling) and erythema (redness) before and for up to 15 days after injection. Draize scoring was used to score sites based on the severity of the response (maximum 0-4). No abnormal findings were observed in any of the five animals in the composition groups (100 mg / kg, 500 mg / mL) from day 0 (pre-injection) to day 14 (post-injection).

[0300] Erythema and crusting: No erythema - 0; Very mild erythema (barely perceptible) - 1; Well-defined erythema - 2; Moderate to severe erythema - 3; Severe erythema (beef redness) to crusting that interferes with erythema grading - 4.

[0301] Edema formation: No edema - 0; Very mild edema (barely perceptible) - 1; Mild edema (border of area clearly defined by a distinct elevation) - 2; Moderate edema (elevation of approximately 1 mm) - 3; Severe edema (elevation greater than 1 mm and expansion beyond the exposed area) - 4.

[0302] Draize scoring: The presence or absence of findings was recorded for each individual animal from day 0 (pre-injection) to day 14 (post-injection). All five animals in the group had zero edema and erythema scores over 14 days post-injection, indicating that the composition sample was well-tolerated without toxicity.

[0303] Detailed clinical observations: Animals were removed from their cages and observed. These included, but were not limited to, evaluations of the effects on the skin, fur, eyes, ears, nose, mouth, chest, abdomen, genitals, limbs and feet, respiratory and circulatory systems, autonomic nervous system effects such as salivation, and nervous system effects including tremors, convulsions, responsiveness to handling, and abnormal behavior. No abnormal findings were observed in any of the five animals in the composition groups (100 mg / kg, 500 mg / mL) from day 0 (before injection) to day 14 (after injection), indicating that the composition samples were well tolerated without observable toxicity.

[0304] Local tolerability read from histopathology of injection sites in miniature pigs showed minimal to mild changes with and without hyaluronidase in both aqueous and suspension formulations, with and without adverse effects (Figures 18A-F). Addition of hyaluronidase to the formulations did not significantly affect the injection site. Similar to the aqueous formulations, the suspension formulations did not cause any adverse reactions at the injection site.

[0305] Metabolic and CBC panels containing 25 serum analytes were performed on miniature pigs on days 4, 8, and 15 after injection (Figure 19A-B). The results showed that injection of PGD and hyaluronidase-containing suspensions over a two-week period did not significantly alter any markers, and no systemic toxicity was observed.

[0306] Figure 20 shows that adding hyaluronidase to microparticles increases the bioavailability of the suspension in Sprague Dawley rats. Figure 20 shows that the sample with hyaluronidase (red trace) showed increased plasma concentrations of the therapeutic protein trastuzumab compared to the formulation without hyaluronidase (blue trace). The increases in AUC and cMax (pharmacokinetic parameters) for the aqueous f...

Claims

1. A particle comprising at least one therapeutic biological agent or a salt thereof, and a hyaluronan degrading agent.

2. The particle according to claim 1, wherein the hyaluronan degrading agent is hyaluronidase.

3. The particle according to claim 2, wherein the hyaluronidase is a soluble neutral-active hyaluronidase.

4. The particle according to claim 2, wherein the hyaluronidase is mammalian hyaluronidase.

5. The particle according to claim 4, wherein the mammalian hyaluronidase is human hyaluronidase.

6. The particle according to claim 5, wherein the human hyaluronidase is recombinant human hyaluronidase (rHuPH20).

7. The particle according to any one of the prior claims, wherein the amount of the hyaluronan degrading agent in the particle is less than about 1% by weight.

8. The particle according to any one of the prior claims, wherein the hyaluronan degrading agent in the particle retains at least 50% of the activity of an effective amount of the hyaluronan degrading agent before particle formation.

9. The particles according to any one of the prior claims, wherein the particles have a coagulation of less than about 10% of the therapeutic biological preparation.

10. The particles according to any one of the prior claims, wherein the particles substantially do not contain any of the coagulations of the therapeutic biological preparation.

11. The particles according to any one of the prior claims, wherein the particles have less than 10% fragmentation of the therapeutic biological preparation.

12. The particles according to any one of the prior claims, wherein the particles substantially do not contain any fragmentation of the therapeutic biological preparation.

13. The particle according to any one of the prior claims, wherein the particle has a change of less than about 5% in the charge variant of the therapeutic biological agent.

14. The particles according to any one of the prior claims, wherein the particles substantially do not include any changes in the charge variant of the therapeutic biological agent.

15. The particle according to any one of the prior claims, wherein the particle has a roundness of about 0.8 to about 1.

00.

16. The particles according to any one of the prior claims, wherein the particles have less than about 7% by weight of water.

17. The particle according to any one of the prior claims, wherein the particle has an internal void space of less than about 25%.

18. The particle according to any one of the prior claims, wherein the particle substantially does not contain any internal void space.

19. The particle according to any one of the prior claims, wherein the particle comprises more than 60% by weight of a therapeutic biological preparation.

20. The particles according to any one of the prior claims, wherein the particles further comprise a carbohydrate, a pH adjuster, a salt, a chelating agent, a surfactant, a protein stabilizer, an emulsifier, a preservative, an amino acid, an antioxidant, a paraben, a bactericide, a fungicide, a preservative, or a combination thereof.

21. The particles according to claim 20, wherein the carbohydrate is dextran, trehalose, sucrose, agarose, mannitol, lactose, sorbitol, maltose, or a combination thereof.

22. The particles according to claim 20, wherein the pH adjusting agent is acetate, citrate, glutamate, glycinate, histidine, lactate, maleate, phosphate, succinate, tartrate, bicarbonate, aluminum hydroxide, phosphoric acid, hydrochloric acid, DL-lactic acid / glycolic acid, phosphorylethanolamine, tromethamine, imidazole, glycryglycine, monosodium glutamate, sodium hydroxide, potassium hydroxide, or a combination thereof.

23. The particles according to claim 20, wherein the salt is sodium chloride, calcium chloride, potassium chloride, sodium hydroxide, stannous chloride, magnesium sulfate, sodium glucoheptonate, sodium pertechnetate, guanidine hydrochloride, potassium hydroxide, magnesium chloride, potassium nitrate, or a combination thereof.

24. The particles according to claim 20, wherein the chelating agent is disodium edetate, ethylenediaminetetraacetate pentetate, or a combination thereof.

25. The particles according to claim 20, wherein the surfactant is sorbitan ester, polysorbate, poloxamer, polyethylene glycol tert-octylphenyl ether, lecithin, sodium stearate, magnesium stearate, or a combination thereof.

26. The particles according to claim 20, wherein the protein stabilizer is trehalose, polyethylene glycol (PEG), polyoxomer, polyvinylpyrrolidone, polyacrylic acid, poly(vinyl) polymer, polyester, polyaldehyde, tert-polymer, polyamino acid, hydroxyethyl starch, N-methyl-2-pyrrolidone, sorbitol, sucrose, mannitol, cyclodextrin, hydroxypropyl beta-cyclodextrin, sulfobutyl ether beta-cyclodextrin, methyl beta-cyclodextrin, acetyltryptophanate, caprylate, N-acetyltryptophan, propylene glycol, glucose star polymer, silicone polymer, polydimethylsiloxane, carboxymethylcellulose, poly(glycolic acid), poly(lactic acid-coglycolic acid), polylactic acid, polycaprolactone (PCL), polyvinylpyrrolidone (PVP), Ficol, dextran, or a combination thereof.

27. The particles according to claim 20, wherein the emulsifier is polysorbate, sorbitan monooleate, ethanolamine, polyoxyl 35 castor oil, poloxyl 40 hydrogenated castor oil, carbomer 1342, corn oil mono-di-triglyceride, polyoxyethylated oleiceride, poloxamer, or a combination thereof.

28. The particles according to claim 20, wherein the preservative is phenol, m-cresol, benzyl alcohol, 2-phenyloxyethanol, chlorobutanol, neomycin, benzethonium chloride, gluteraldehyde, beta-propiolactone, or a combination thereof.

29. The particle according to claim 20, wherein the amino acid is alanine, aspartic acid, cysteine, isoleucine, glutamic acid, leucine, methionine, phenylalanine, pyrrolicin, serine, selenocysteine, threonine, tryptophan, tyrosine, valine, asparagine, arginine, histidine, glycine, glutamine, proline, or a combination thereof.

30. The particles according to claim 20, wherein the antioxidant is glutathione, ascorbic acid, cysteine, N-acetyl-L-tryptophanate, tocopherol, histidine, methionine, pentetic acid, or a combination thereof.

31. The particle according to claim 20, wherein the paraben is parahydroxybenzoic acid.

32. The particles according to claim 20, wherein the disinfectant is benzalkonium chloride.

33. The particles according to claim 20, wherein the fungicide is acibenzoral, 2-phenylphenol, anilazine, carvone, natamycin, potassium azide, or a combination thereof.

34. The particles according to claim 20, wherein the preservative is sodium nitrate, sulfur dioxide, potassium sorbate, sodium sorbate, sodium benzoate, benzoic acid, methyl hydroxybenzoate, thimerosal, parabens, formaldehyde, castor oil, or a combination thereof.

35. The particle according to claim 20, wherein the particle comprises at least carbohydrates and amino acids.

36. The particle according to claim 20, wherein the particle comprises at least a surfactant and an amino acid.

37. The particle according to claim 20, wherein the particle comprises at least an amino acid.

38. The particle according to any one of the prior claims, wherein the therapeutic biological preparation is an antibody or a fragment thereof.

39. The particle according to claim 38, wherein the antibody is a human antibody.

40. The particle according to claim 39, wherein the human antibody is an IgG antibody.

41. The particle according to claim 38, wherein the antibody is a monoclonal antibody.

42. The particle according to claim 41, wherein the monoclonal antibody is rituximab or trastuzumab.

43. The particle according to any one of claims 1 to 37, wherein the therapeutic biological preparation is BSA.

44. The particle according to any one of claims 1 to 43, wherein the particle comprises at least two therapeutic biological agents or salts thereof.

45. The particles according to any one of the prior claims, wherein when the particles are dissolved in an aqueous liquid, they substantially do not contain visible particles (VP).

46. The particles according to any one of the prior claims, wherein when the particles are dissolved in an aqueous liquid, the concentration of insoluble sub-visible particles (SvP) having a characteristic size greater than about 10 μm is about 0 to about 100,000,000 per 1 mL.

47. The particles according to any one of the prior claims, wherein when the particles are dissolved in an aqueous liquid, the concentration of insoluble sub-visible particles (SvP) having a characteristic size greater than about 10 μm is about 0 to about 6000 per 1 mL.

48. The particles according to any one of the prior claims, wherein when the particles are dissolved in an aqueous liquid, they have a concentration of about 0 to about 600 per mL as insoluble sub-visible particles (SvP) having a characteristic size greater than about 25 μm.

49. The particles according to any one of the prior claims, wherein when dissolved in an aqueous liquid, the particles substantially contain no insoluble sub-visible particles (SvP).

50. The particles according to any one of the prior claims, wherein the aqueous liquid is water, an aqueous buffer solution, or a physiologically related aqueous liquid.

51. The particles according to any one of the prior claims, wherein the hyaluronan degrading agent has improved stability compared to an aqueous composition containing the hyaluronan degrading agent in a soluble form.

52. The particle according to any one of the prior claims, wherein the therapeutic biological agent and the hyaluronan degrading agent in the particle are stable for at least one month.

53. The particle according to any one of the prior claims, wherein the therapeutic biological agent and the hyaluronan degrading agent in the particle are stable for at least three months.

54. The particles according to any one of the prior claims, wherein the therapeutic biological agent and the hyaluronan degrading agent in the particles are stable at 40°C for at least three months.

55. A pharmaceutically effective composition, A pharmaceutically effective composition comprising a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein substantially all of the particles comprise at least one therapeutic biological agent or a salt thereof and a hyaluronan degrader.

56. The composition according to claim 55, wherein the concentration of the hyaluronan degrading agent in the composition is less than about 20,000 U / mL.

57. The composition according to claim 55 or 56, wherein the hyaluronan degrading agent is hyaluronidase.

58. The composition according to claim 57, wherein the hyaluronidase is a soluble neutral-active hyaluronidase.

59. The composition according to claim 57, wherein the hyaluronidase is mammalian hyaluronidase.

60. The composition according to claim 59, wherein the mammalian hyaluronidase is human hyaluronidase.

61. The composition according to claim 60, wherein the human hyaluronidase is recombinant human hyaluronidase (rHuPH20).

62. The composition according to any one of claims 55 to 61, wherein substantially all of the particles comprise at least one therapeutic biological agent and a hyaluronan degrading agent.

63. The composition according to any one of claims 55 to 62, wherein substantially all of the particles have a coagulation of less than about 10% of the therapeutic biological preparation.

64. The composition according to any one of claims 55 to 63, wherein substantially all of the particles substantially do not contain any of the coagulations of the therapeutic biological preparation.

65. The composition according to any one of claims 55 to 64, wherein substantially all of the particles have less than about 10% fragmentation of the therapeutic biological preparation.

66. The composition according to any one of claims 55 to 65, wherein substantially all of the particles substantially do not contain any fragmentation of the therapeutic biological preparation.

67. The composition according to any one of claims 55 to 66, wherein substantially all of the particles have a change of less than about 5% in the charge variant of the therapeutic biological agent.

68. The composition according to any one of claims 55 to 67, wherein substantially all of the particles substantially do not contain any changes in the charge variant of the therapeutic biological agent.

69. The composition according to any one of claims 55 to 68, wherein substantially all of the particles have a roundness of about 0.80 to about 1.

00.

70. The composition according to any one of claims 55 to 69, wherein substantially all of the particles have a roundness of about 1.

00.

71. The composition according to any one of claims 55 to 70, wherein substantially all of the particles have less than about 7% by weight of water.

72. The composition according to any one of claims 55 to 71, wherein substantially all of the particles have an internal void space of less than about 25%.

73. The composition according to any one of claims 55 to 72, wherein substantially all of the particles substantially do not contain any internal void spaces.

74. The composition according to any one of claims 55 to 73, wherein substantially all of the particles contain more than 60% by weight of a therapeutic biological agent.

75. The composition according to any one of claims 55 to 74, wherein substantially all of the particles further comprise carbohydrates, pH adjusters, salts, chelating agents, surfactants, protein stabilizers, emulsifiers, preservatives, amino acids, antioxidants, parabens, bactericides, fungicides, preservatives, or combinations thereof.

76. The composition according to claim 75, wherein the carbohydrate is dextran, trehalose, sucrose, agarose, mannitol, lactose, sorbitol, maltose, or a combination thereof.

77. The composition according to claim 75, wherein the pH adjusting agent is an acetate, citrate, glutamate, glycinate, histidine, lactate, maleate, phosphate, succinate, tartrate, bicarbonate, aluminum hydroxide, phosphoric acid, hydrochloric acid, DL-lactic acid / glycolic acid, phosphorylethanolamine, tromethamine, imidazole, glycryglycine, monosodium glutamate, sodium hydroxide, potassium hydroxide, or a combination thereof.

78. The composition according to claim 75, wherein the salt is sodium chloride, calcium chloride, potassium chloride, sodium hydroxide, stannous chloride, magnesium sulfate, sodium glucoheptonate, sodium pertechnetate, guanidine hydrochloride, potassium hydroxide, magnesium chloride, potassium nitrate, or a combination thereof.

79. The composition according to claim 75, wherein the chelating agent is disodium edetate, pentetic acid ethylenediaminetetraacetate, or a combination thereof.

80. The composition according to claim 75, wherein the surfactant is sorbitan ester, polysorbate, poloxamer, polyethylene glycol tert-octylphenyl ether, lecithin, sodium stearate, magnesium stearate, or a combination thereof.

81. The composition according to claim 75, wherein the protein stabilizer is trehalose, polyethylene glycol (PEG), polyoxomer, polyvinylpyrrolidone, polyacrylic acid, poly(vinyl) polymer, polyester, polyaldehyde, tert-polymer, polyamino acid, hydroxyethyl starch, N-methyl-2-pyrrolidone, sorbitol, sucrose, mannitol, cyclodextrin, hydroxypropyl beta-cyclodextrin, sulfobutyl ether beta-cyclodextrin, methyl beta-cyclodextrin, acetyltryptophanate, caprylate, N-acetyltryptophan, propylene glycol, glucose star polymer, silicone polymer, polydimethylsiloxane, carboxymethylcellulose, poly(glycolic acid), poly(lactic acid-coglycolic acid), polylactic acid, polycaprolactone (PCL), polyvinylpyrrolidone (PVP), Ficol, dextran, or a combination thereof.

82. The composition according to claim 75, wherein the emulsifier is polysorbate, sorbitan monooleate, ethanolamine, polyoxyl 35 castor oil, poloxyl 40 hydrogenated castor oil, carbomer 1342, corn oil mono-di-triglyceride, polyoxyethylated oleiceride, poloxamer, or a combination thereof.

83. The composition according to claim 75, wherein the preservative is phenol, m-cresol, benzyl alcohol, 2-phenyloxyethanol, chlorobutanol, neomycin, benzethonium chloride, gluteraldehyde, beta-propiolactone, or a combination thereof.

84. The composition according to claim 75, wherein the amino acid is alanine, aspartic acid, cysteine, isoleucine, glutamic acid, leucine, methionine, phenylalanine, pyrrolicin, serine, selenocysteine, threonine, tryptophan, tyrosine, valine, asparagine, arginine, histidine, glycine, glutamine, proline, or a combination thereof.

85. The composition according to claim 75, wherein the antioxidant is glutathione, ascorbic acid, cysteine, N-acetyl-L-tryptophanate, tocopherol, histidine, methionine, pentetic acid, or a combination thereof.

86. The composition according to claim 75, wherein the paraben is parahydroxybenzoic acid.

87. The composition according to claim 75, wherein the disinfectant is benzalkonium chloride.

88. The composition according to claim 75, wherein the fungicide is acibenzoral, 2-phenylphenol, anilazine, carvone, natamycin, potassium azide, or a combination thereof.

89. The composition according to claim 75, wherein the preservative is sodium nitrate, sulfur dioxide, potassium sorbate, sodium sorbate, sodium benzoate, benzoic acid, methyl hydroxybenzoate, thimerosal, parabens, formaldehyde, castor oil, or a combination thereof.

90. The composition according to claim 75, wherein substantially all of the particles comprise at least carbohydrates and amino acids.

91. The composition according to claim 75, wherein substantially all of the particles comprise at least a surfactant and an amino acid.

92. The composition according to claim 75, wherein substantially all of the particles contain at least an amino acid.

93. The composition according to any one of claims 55 to 92, wherein the therapeutic biological preparation is an antibody or a fragment thereof.

94. The composition according to claim 93, wherein the antibody is a human antibody.

95. The composition according to claim 94, wherein the human antibody is an IgG antibody.

96. The composition according to claim 93, wherein the antibody is a monoclonal antibody.

97. The composition according to claim 96, wherein the monoclonal antibody is rituximab or trastuzumab.

98. The composition according to any one of claims 55 to 92, wherein the therapeutic biological agent is BSA.

99. The composition according to any one of claims 55 to 98, wherein the particles comprise at least two therapeutic biological agents or salts thereof.

100. The composition according to any one of claims 55 to 99, wherein the liquid carrier is non-aqueous.

101. The composition according to claim 100, wherein the non-aqueous liquid carrier is an organic solvent.

102. The organic solvents mentioned above include benzyl benzoate, coconut oil, cottonseed oil, fish oil, grape seed oil, hazelnut oil, hydrogenated vegetable oil, olive oil, coconut seed oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, sunflower oil, walnut oil, corn oil, acetone, ethyl acetate, ethyl lactate, dimethylacetamide, dimethyl isosorbide, dimethyl sulfoxide, glycoflor, diglym, methyl tert-butyl ether, N-methylpyrrolidone, perfluorodecalin, polyethylene glycol, 2-pyrrolidone, and tetrahydrofurfuryl alcohol. Diglycerides, triglycerides, medium-chain triglycerides (MCTs), caproic acid, caprylic acid, capric acid, lauric acid, ethyl laureate, triglycerides of fractionated C8 and C10 vegetable fatty acids, propylene glycol diesters (PGD) of saturated C8 and C10 vegetable fatty acids, ethyl oleate, ethyl caprate, dibutyl adipate, fatty acid esters, hexanoic acid, octanoic acid, triacetin, diethyl glycol monoether, gamma-butyrolactone, eugenol, clove bud oil, citral, limonene, polyoxyl 40 hydrogenated castor oil Oil, polyoxyl 35 castor oil, simple alcohols, e.g., ethanol, octanol, hexanol, decanol, propanol, butanol, gamma-butyrolactone, tocopherol, octa-fluoropropane, (perfluorohexyl)octane, n-acetyltryptophan, ethyl laurate, methyl caprylate, methyl caprate, methyl myristate, methyl oleate, methyl linoleate, dimethyl adipate, dibutyl suberate, diethyl sebacate, ethyl macadamiate, triisostearyl ester Trimethylolpropane phosphate, isopropyl laurate, isopropyl myristate, diethyl succinate, polysorbate ester, ethanolamine, propanoic acid, citral, anisole, anethole, benzaldehyde, linalool, caprolactone, phenol, thioglycerol, dimethylacetamide, diethylene glycol monoethyl ether, solketal, isosorbide dimethyl ether, ethyl formate, hexyl ethyl acetate, propylene glycol dicaprylate, caprylic acid triglyceride, ethyl linoleate, ethyl linolenate,The composition according to claim 101, or a combination thereof.

103. The composition according to claim 101, wherein the organic solvent is ethyl oleate, diglycerides, triglycerides, ethyl macadamia nut fatty acid, ethyl caprate, diethyl succinate, diethylene glycol monoethyl ether, propylene glycol dicaprylate, caprylic acid triglyceride, ethyl linoleate, ethyl linolenate, medium-chain triglycerides (MCT), sesame oil, propylene glycol diesters (PGD) of saturated vegetable fatty acids C8 and C10, triacetin, or a combination thereof.

104. The composition according to claim 101, wherein the organic solvent is ethyl oleate, ethyl caprylate, propylene glycol dicaprylate, diglycerides, triglycerides, sesame oil, propylene glycol diesters (PGD) of saturated vegetable fatty acids C8 and C10, triacetin, or a combination thereof.

105. The composition according to claim 100, wherein the non-aqueous liquid carrier comprises at least two organic solvents.

106. The composition according to any one of claims 55 to 105, wherein the composition further comprises a flocculant.

107. The composition according to claim 106, wherein the concentration of the flocculant in the composition is less than about 50 mg / mL.

108. The composition according to claim 106, wherein the flocculant is polysorbate, alkylphenol ethoxylate, glycerol, polyoxyethylated castor oil, doxate, decyl glucoside, nonoxynol-9, sorbitan ester, sorbitan monooleate, ethanolamine, polyoxyl 35 castor oil, poloxyl 40 hydrogenated castor oil, carbomer 1342, corn oil mono-di-triglyceride, polyoxyethylated oleiceride, poloxamer, or a combination thereof.

109. The composition according to any one of claims 55 to 108, wherein the composition has an aggregate volume of more than about 60% after initial mixing.

110. The composition according to any one of claims 55 to 109, wherein the composition has substantially the same aggregate volume for at least one month under conditions of closed storage in a container at less than about 40°C.

111. The composition according to any one of claims 55 to 110, wherein the concentration of the therapeutic biological agent in the composition is greater than about 250 mg / mL.

112. The composition according to any one of claims 55 to 111, wherein the composition has a viscosity of less than about 100 mPa·s.

113. The composition according to any one of claims 55 to 112, wherein when the composition is dissolved in an aqueous liquid, it is substantially free of visible particles (VP).

114. The composition according to any one of claims 55 to 113, wherein when the composition is dissolved in an aqueous liquid, the concentration of insoluble sub-visible particles (SvP) having a characteristic size greater than about 10 μm is about 0 to about 100,000,000 per 1 mL.

115. The composition according to any one of claims 55 to 114, wherein when the composition is dissolved in an aqueous liquid, the concentration of insoluble sub-visible particles (SvP) having a characteristic size greater than about 10 μm is about 0 to about 6000 per 1 mL.

116. The composition according to any one of claims 55 to 115, wherein when the composition is dissolved in an aqueous liquid, the concentration of insoluble sub-visible particles (SvP) having a characteristic size greater than about 25 μm is about 0 to about 600 per 1 mL.

117. The composition according to any one of claims 55 to 116, wherein when the composition is dissolved in an aqueous liquid, it is substantially free of insoluble sub-visible particles (SvP).

118. The composition according to any one of claims 55 to 117, wherein the aqueous liquid is water, an aqueous buffer solution, or a physiologically related aqueous liquid.

119. The composition according to any one of claims 55 to 118, wherein the composition has improved pharmacokinetics (PK) compared to a composition comprising a plurality of particles each comprising at least one therapeutic biological agent.

120. The composition according to any one of claims 55 to 119, wherein the hyaluronan degrading agent has improved stability compared to an aqueous composition containing the hyaluronan degrading agent in a soluble form.

121. The composition according to any one of claims 55 to 120, wherein the therapeutic biological agent and the hyaluronan degrading agent in the composition are stable for at least one month.

122. The composition according to any one of claims 55 to 121, wherein the therapeutic biological agent and the hyaluronan degrading agent in the composition are stable for at least three months.

123. The composition according to any one of claims 55 to 122, wherein the therapeutic biological agent and the hyaluronan degrading agent in the composition are stable at 40°C for at least three months.

124. A method of treating a disease or condition in a person who requires treatment for a disease or condition, A method comprising administering to a subject a pharmaceutically effective amount of a composition comprising a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein substantially all of the particles comprise at least one therapeutic biological agent or a salt thereof and a hyaluronan degrader.

125. The method according to claim 124, wherein the concentration of the hyaluronan degrading agent in the composition is less than about 20,000 U / mL.

126. The method according to claim 124 or 125, wherein the hyaluronan decomposing agent is hyaluronidase.

127. The method according to claim 126, wherein the hyaluronidase is a soluble neutral-active hyaluronidase.

128. The method according to claim 126, wherein the hyaluronidase is mammalian hyaluronidase.

129. The method according to claim 128, wherein the mammalian hyaluronidase is human hyaluronidase.

130. The method according to claim 129, wherein the human hyaluronidase is recombinant human hyaluronidase (rHuPH20).

131. The method according to any one of claims 124 to 130, wherein substantially all of the particles comprise at least one therapeutic biological agent.

132. The method according to any one of claims 124 to 131, wherein the disease or condition is cancer, an inflammatory disease, or an immune disease.

133. The method according to any one of claims 124 to 132, wherein substantially all of the particles comprise at least one therapeutic biological agent and a hyaluronan degrading agent.

134. The method according to any one of claims 124 to 133, wherein substantially all of the particles have a coagulation of less than about 10% of the therapeutic biological formulation.

135. The method according to any one of claims 124 to 134, wherein substantially all of the particles substantially do not contain any of the coagulations of the therapeutic biological preparation.

136. The method according to any one of claims 124 to 135, wherein substantially all of the particles have less than about 10% fragmentation of the therapeutic biological formulation.

137. The method according to any one of claims 124 to 136, wherein substantially all of the particles substantially do not contain any fragmentation of the therapeutic biological preparation.

138. The method according to any one of claims 124 to 137, wherein substantially all of the particles have a change of less than about 5% in the charge variant of the therapeutic biological agent.

139. The method according to any one of claims 124 to 138, wherein substantially all of the particles substantially do not contain any changes in the charge variant of the therapeutic biological agent.

140. The method according to any one of claims 124 to 139, wherein substantially all of the particles have a roundness of about 0.80 to about 1.

00.

141. The method according to any one of claims 124 to 140, wherein substantially all of the particles have a roundness of about 1.

00.

142. The method according to any one of claims 124 to 141, wherein substantially all of the particles have less than about 7% by weight of water.

143. The method according to any one of claims 124 to 142, wherein substantially all of the particles have an internal void space of less than about 25%.

144. The method according to any one of claims 124 to 143, wherein substantially all of the particles substantially do not contain any internal void spaces.

145. The method according to any one of claims 124 to 144, wherein substantially all of the particles contain more than 60% by weight of a therapeutic biological agent.

146. The method according to any one of claims 124 to 145, wherein substantially all of the particles further comprise carbohydrates, pH adjusters, salts, chelating agents, surfactants, protein stabilizers, emulsifiers, preservatives, amino acids, antioxidants, parabens, bactericides, fungicides, preservatives, or combinations thereof.

147. The method according to claim 146, wherein the carbohydrate is dextran, trehalose, sucrose, agarose, mannitol, lactose, sorbitol, maltose, or a combination thereof.

148. The method according to claim 146, wherein the pH adjusting agent is an acetate, citrate, glutamate, glycinate, histidine, lactate, maleate, phosphate, succinate, tartrate, bicarbonate, aluminum hydroxide, phosphoric acid, hydrochloric acid, DL-lactic acid / glycolic acid, phosphorylethanolamine, tromethamine, imidazole, glycylglycine, monosodium glutamate, sodium hydroxide, potassium hydroxide, or a combination thereof.

149. The method according to claim 146, wherein the salt is sodium chloride, calcium chloride, potassium chloride, sodium hydroxide, stannous chloride, magnesium sulfate, sodium glucoheptonate, sodium pertechnetate, guanidine hydrochloride, potassium hydroxide, magnesium chloride, potassium nitrate, or a combination thereof.

150. The method according to claim 146, wherein the chelating agent is disodium edetate, pentetic acid ethylenediaminetetraacetate, or a combination thereof.

151. The method according to claim 146, wherein the surfactant is sorbitan ester, polysorbate, poloxamer, polyethylene glycol tert-octylphenyl ether, lecithin, sodium stearate, magnesium stearate, or a combination thereof.

152. The method according to claim 146, wherein the protein stabilizer is trehalose, polyethylene glycol (PEG), polyoxomer, polyvinylpyrrolidone, polyacrylic acid, poly(vinyl) polymer, polyester, polyaldehyde, tert-polymer, polyamino acid, hydroxyethyl starch, N-methyl-2-pyrrolidone, sorbitol, sucrose, mannitol, cyclodextrin, hydroxypropyl beta-cyclodextrin, sulfobutyl ether beta-cyclodextrin, acetyltryptophanate, methyl beta-cyclodextrin, caprylate, N-acetyltryptophan, propylene glycol, glucose star polymer, silicone polymer, polydimethylsiloxane, carboxymethylcellulose, poly(glycolic acid), poly(lactic acid-coglycolic acid), polylactic acid, polycaprolactone (PCL), polyvinylpyrrolidone (PVP), Ficol, dextran, or a combination thereof.

153. The method according to claim 146, wherein the emulsifier is polysorbate, sorbitan monooleate, ethanolamine, polyoxyl 35 castor oil, poloxyl 40 hydrogenated castor oil, carbomer 1342, corn oil mono-di-triglyceride, polyoxyethylated oleiceride, poloxamer, or a combination thereof.

154. The method according to claim 146, wherein the preservative is phenol, m-cresol, benzyl alcohol, 2-phenyloxyethanol, chlorobutanol, neomycin, benzethonium chloride, gluteraldehyde, beta-propiolactone, or a combination thereof.

155. The method according to claim 146, wherein the amino acid is alanine, aspartic acid, cysteine, isoleucine, glutamic acid, leucine, methionine, phenylalanine, pyrrolicin, serine, selenocysteine, threonine, tryptophan, tyrosine, valine, asparagine, arginine, histidine, glycine, glutamine, proline, or a combination thereof.

156. The method according to claim 146, wherein the antioxidant is glutathione, ascorbic acid, cysteine, N-acetyl-L-tryptophanate, tocopherol, histidine, methionine, pentetic acid, or a combination thereof.

157. The method according to claim 146, wherein the paraben is para-hydroxybenzoic acid.

158. The method according to claim 146, wherein the disinfectant is benzalkonium chloride.

159. The method according to claim 146, wherein the fungicide is acibenzoral, 2-phenylphenol, anilazine, carvone, natamycin, potassium azide, or a combination thereof.

160. The method according to claim 146, wherein the preservative is sodium nitrate, sulfur dioxide, potassium sorbate, sodium sorbate, sodium benzoate, benzoic acid, methyl hydroxybenzoate, thimerosal, parabens, formaldehyde, castor oil, or a combination thereof.

161. The method according to claim 146, wherein substantially all of the particles comprise at least carbohydrates and amino acids.

162. The method according to claim 146, wherein substantially all of the particles comprise at least a surfactant and an amino acid.

163. The method according to claim 146, wherein substantially all of the particles contain at least an amino acid.

164. The method according to any one of claims 124 to 163, wherein the therapeutic biological preparation is an antibody or a fragment thereof.

165. The method according to claim 164, wherein the antibody is a human antibody.

166. The method according to claim 165, wherein the human antibody is an IgG antibody.

167. The method according to claim 164, wherein the antibody is a monoclonal antibody.

168. The method according to claim 167, wherein the monoclonal antibody is rituximab or trastuzumab.

169. The method according to any one of claims 124 to 163, wherein the therapeutic biological agent is BSA.

170. The method according to any one of claims 124 to 169, wherein the particles comprise at least two therapeutic biological agents or salts thereof.

171. The method according to any one of claims 124 to 170, wherein the liquid carrier is non-aqueous.

172. The method according to claim 171, wherein the non-aqueous liquid carrier is an organic solvent.

173. The organic solvents mentioned above include benzyl benzoate, coconut oil, cottonseed oil, fish oil, grape seed oil, hazelnut oil, hydrogenated vegetable oil, olive oil, coconut seed oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, sunflower oil, walnut oil, corn oil, acetone, ethyl acetate, ethyl lactate, dimethylacetamide, dimethyl isosorbide, dimethyl sulfoxide, glycoflor, diglym, methyl tert-butyl ether, N-methylpyrrolidone, perfluorodecalin, polyethylene glycol, 2-pyrrolidone, and tetrahydrofurfuryl alcohol. Diglycerides, triglycerides, medium-chain triglycerides (MCTs), caproic acid, caprylic acid, capric acid, lauric acid, ethyl laureate, triglycerides of fractionated C8 and C10 vegetable fatty acids, propylene glycol diesters (PGD) of saturated C8 and C10 vegetable fatty acids, ethyl oleate, ethyl caprate, dibutyl adipate, fatty acid esters, hexanoic acid, octanoic acid, triacetin, diethyl glycol monoether, gamma-butyrolactone, eugenol, clove bud oil, citral, limonene, polyoxyl 40 hydrogenated castor oil Oil, polyoxyl 35 castor oil, simple alcohols, e.g., ethanol, octanol, hexanol, decanol, propanol, butanol, gamma-butyrolactone, tocopherol, octa-fluoropropane, (perfluorohexyl)octane, n-acetyltryptophan, ethyl laurate, methyl caprylate, methyl caprate, methyl myristate, methyl oleate, methyl linoleate, dimethyl adipate, dibutyl suberate, diethyl sebacate, ethyl macadamiate, triisostearyl ester Trimethylolpropane phosphate, isopropyl laurate, isopropyl myristate, diethyl succinate, polysorbate ester, ethanolamine, propanoic acid, citral, anisole, anethole, benzaldehyde, linalool, caprolactone, phenol, thioglycerol, dimethylacetamide, diethylene glycol monoethyl ether, solketal, isosorbide dimethyl ether, ethyl formate, hexyl ethyl acetate, propylene glycol dicaprylate, caprylic acid triglyceride, ethyl linoleate, ethyl linolenate,The method according to claim 172, including a combination thereof.

174. The method according to claim 172, wherein the organic solvent is ethyl oleate, diglycerides, triglycerides, ethyl macadamia nut fatty acid, ethyl caprate, diethyl succinate, diethylene glycol monoethyl ether, propylene glycol dicaprylate, caprylic triglyceride, ethyl linoleate, ethyl linolenate, medium-chain triglycerides (MCT), sesame oil, propylene glycol diesters (PGD) of saturated vegetable fatty acids C8 and C10, triacetin, or a combination thereof.

175. The method according to claim 172, wherein the organic solvent is ethyl oleate, ethyl caprylate, propylene glycol dicaprylate, diglycerides, triglycerides, sesame oil, propylene glycol diesters (PGD) of saturated vegetable fatty acids C8 and C10, triacetin, or a combination thereof.

176. The method according to claim 171, wherein the non-aqueous liquid carrier comprises at least two organic solvents.

177. The method according to any one of claims 124 to 176, wherein the composition further comprises a flocculant.

178. The method according to claim 177, wherein the concentration of the flocculant in the composition is less than about 50 mg / mL.

179. The method according to claim 177, wherein the flocculant is polysorbate, alkylphenol ethoxylate, glycerol, polyoxyethylated castor oil, doxate, decyl glucoside, nonoxynol-9, sorbitan ester, sorbitan monooleate, ethanolamine, polyoxyl 35 castor oil, poloxyl 40 hydrogenated castor oil, carbomer 1342, corn oil mono-di-triglyceride, polyoxyethylated oleiceride, poloxamer, or a combination thereof.

180. The method according to any one of claims 124 to 179, wherein the composition has a coagulation volume of more than about 60% after initial mixing.

181. The method according to any one of claims 124 to 180, wherein the composition has substantially the same aggregate volume for at least one month under conditions of closed storage in a container at less than about 40°C.

182. The method according to any one of claims 124 to 181, wherein the concentration of the therapeutic biological agent in the composition is greater than about 250 mg / mL.

183. The method according to any one of claims 124 to 182, wherein the composition has a viscosity of less than about 100 mPa·s.

184. The method according to any one of claims 124 to 183, wherein the composition, when dissolved in an aqueous liquid, is substantially free of visible particles (VP).

185. The method according to any one of claims 124 to 184, wherein when the composition is dissolved in an aqueous liquid, the concentration of insoluble sub-visible particles (SvP) having a characteristic size greater than about 10 μm is about 0 to about 100,000,000 per mL.

186. The method according to any one of claims 124 to 185, wherein when the composition is dissolved in an aqueous liquid, the concentration of insoluble sub-visible particles (SvP) having a characteristic size greater than about 10 μm is about 0 to about 6000 per mL.

187. The method according to any one of claims 124 to 186, wherein when the composition is dissolved in an aqueous liquid, the concentration of insoluble sub-visible particles (SvP) having a characteristic size greater than about 25 μm is about 0 to about 600 per 1 mL.

188. The method according to any one of claims 124 to 187, wherein the composition, when dissolved in an aqueous liquid, is substantially free of insoluble sub-visible particles (SvP).

189. The method according to any one of claims 124 to 188, wherein the aqueous liquid is water, an aqueous buffer solution, or a physiologically related aqueous liquid.

190. The method according to any one of claims 124 to 189, wherein the composition is administered by subcutaneous injection.

191. The method according to any one of claims 124 to 190, wherein the composition is administered by syringe or portable drug delivery injection device.

192. The method according to claim 191, wherein the syringe or portable drug delivery injection device is pre-filled with the composition.

193. The method according to claim 191, wherein the composition is administered by syringe.

194. The method according to claim 191, wherein the composition is administered by a portable drug delivery injection device.

195. The method according to claim 194, wherein the portable drug delivery injection device is a pen syringe.

196. The method according to claim 194, wherein the portable drug delivery injection device is an automatic injector.

197. The method according to any one of claims 124 to 196, wherein the composition is dispensed from a needle having a gauge in the range of 18 gauge to 33 gauge.

198. The method according to any one of claims 124 to 197, wherein the composition is distributed using an injection force of less than about 70 N.

199. The method according to any one of claims 124 to 198, wherein the injection force remains substantially the same for at least one month under conditions of container storage at less than approximately 40°C.

200. The method according to any one of claims 124 to 199, wherein the amount of composition administered is less than about 20.0 mL.

201. The method according to any one of claims 124 to 200, wherein the amount of composition administered is less than about 2.0 mL.

202. The method according to any one of claims 124 to 201, wherein the amount of composition administered is less than about 0.5 mL.

203. The method according to any one of claims 124 to 202, wherein the composition dissolves in less than 10 minutes after administration.

204. The method according to any one of claims 124 to 203, wherein the composition dissolves immediately after administration.

205. The method according to any one of claims 124 to 204, wherein the composition is administered in one or more doses.

206. The method according to claim 205, wherein the composition is administered in a single dose.

207. The method according to claim 205, wherein the composition is administered in multiple doses.

208. The method according to any one of claims 124 to 207, wherein the subject is a mammal.

209. The method according to any one of claims 124 to 208, wherein the subject is a human.

210. The method according to any one of claims 124 to 209, wherein the hyaluronan degrading agent has improved stability compared to an aqueous composition containing the hyaluronan degrading agent in a soluble form.

211. The method according to any one of claims 124 to 210, wherein the therapeutic biological preparation in the composition is stable for at least one month.

212. The method according to any one of claims 124 to 211, wherein the therapeutic biological preparation in the composition is stable for at least three months.

213. The method according to any one of claims 124 to 212, wherein the therapeutic biological preparation in the composition is stable at 40°C for at least three months.

214. A pharmaceutically effective composition, A method for administering a pharmaceutically effective composition comprising a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein substantially all of the particles comprise at least one therapeutic biological agent or a salt thereof and a hyaluronan degrader.

215. The method according to claim 214, wherein the composition is administered by subcutaneous injection.

216. The method according to claim 214 or 215, wherein the composition is administered by syringe or portable drug delivery injection device.

217. The method according to claim 216, wherein the syringe or portable drug delivery injection device is pre-filled with the composition.

218. The method according to claim 216, wherein the composition is administered by syringe.

219. The method according to claim 216, wherein the composition is administered by a portable drug delivery injection device.

220. The method according to claim 219, wherein the portable drug delivery injection device is a pen syringe.

221. The method according to claim 219, wherein the portable drug delivery injection device is an automatic injector.

222. The method according to any one of claims 214 to 221, wherein the composition is dispensed from a needle having a gauge in the range of 18 gauge to 33 gauge.

223. The method according to any one of claims 214 to 222, wherein the composition is distributed using an injection force of less than about 70 N.

224. A kit comprising a syringe or portable drug delivery injection device, and a composition, A kit comprising a composition comprising a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein substantially all of the particles comprise at least one therapeutic biological agent or a salt thereof and a hyaluronan degrader.

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