Injectable Suspension

Abstract

The present disclosure provides highly concentrated injectable particle suspensions of therapeutic biologics that allow for administration without the need for resuspension. In particular, the compositions disclosed herein are highly concentrated, low volume injectable particle suspensions that do not require manual premixing or resuspension prior to administration.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 336,743, filed April 29, 2022, the entire teachings of which are incorporated herein by reference.

[0002] The present disclosure relates to compositions and methods that are useful for the delivery (e.g., intramuscular or subcutaneous delivery) of biopharmaceutical products for therapy. In particular, the compositions and methods disclosed herein are high concentration, low volume, and low syringe force injectable particle suspensions of therapeutic biologics that can be administered to patients without the need for resuspension prior to injection. [Background technology]

[0003] Biologics, particularly antibodies, have driven a paradigm shift in the drug discovery and development process over the past few decades, helping patients for whom few or no treatment options previously existed. For example, current monoclonal antibody (mAb) therapies often require large doses administered by intravenous (IV) infusion at high volumes and low concentrations, which can take hours to deliver, causing patient discomfort and increasing the risk of infection. Subcutaneous (SC) injections offer a more desirable alternative for delivery because they reduce the burden on hospital and clinical facilities, require less time, and lower the risk of complications. However, SC injections of mAb particles often require low delivery volumes that necessitate high concentrations that are difficult to obtain. The requirement of high particle concentrations at low delivery volumes can also lead to excessively high injection forces and result in particle settling that may require manual agitation for particle resuspension. Thus, there is a need for highly concentrated, low volume injectable particle compositions that do not require manual premixing or resuspension prior to administration. Summary of the Invention

[0004] Provided herein is a pharma- ceutical effective composition comprising: a plurality of particles suspended in a pharma- ceutically acceptable liquid carrier, the plurality of particles (e.g., substantially all of the particles) comprising at least one therapeutic biological agent or salt thereof; a flocculant, wherein the concentration of the flocculant in the composition is less than about 50 mg / mL; Compositions are provided, wherein the concentration of the therapeutic biologic or salt thereof in the composition is greater than about 250 mg / mL.

[0005] In one aspect, the disclosure provides a method of treating a disease or condition in a subject in need thereof, comprising administering to the subject a pharma- tically effective amount of a composition comprising: a plurality of particles suspended in a pharma- ceutically acceptable liquid carrier, the plurality of particles (e.g., substantially all of the particles) comprising at least one therapeutic biological agent or salt thereof; a flocculant, wherein the concentration of the flocculant in the composition is less than about 50 mg / mL; The method includes administering a composition, wherein the concentration of the therapeutic biologic or a salt thereof in the composition is greater than about 250 mg / mL.

[0006] The disclosure also provides herein a pharma- ceutical effective composition comprising: a plurality of particles suspended in a pharma- ceutically acceptable liquid carrier, the plurality of particles (e.g., substantially all of the particles) comprising at least one therapeutic biological agent or salt thereof; a flocculant, wherein the concentration of the flocculant in the composition is less than about 50 mg / mL; Methods of administering a composition are provided, wherein the concentration of the therapeutic biologic or salt thereof in the composition is greater than about 250 mg / mL.

[0007] In another aspect, the present disclosure provides a pharma- ceutically effective composition comprising particles suspended in a pharma- ceutically acceptable liquid carrier, the particles comprising: a first plurality of particles comprising a first therapeutic biological agent or a salt thereof; particles comprising a second plurality of particles comprising a second therapeutic biological agent or a salt thereof; a flocculant, wherein the concentration of the flocculant in the composition is less than about 50 mg / mL; Compositions are provided, wherein the concentrations of the first and second therapeutic biologics or salts thereof in the composition are greater than about 250 mg / mL.

[0008] The present disclosure also provides a syringe or ambulatory drug delivery injection device and a composition comprising: a plurality of particles suspended in a pharma- ceutically acceptable liquid carrier, the plurality of particles (e.g., substantially all of the particles) comprising at least one therapeutic biological agent or salt thereof; a flocculant, wherein the concentration of the flocculant in the composition is less than about 50 mg / mL; and a composition, wherein the concentration of the therapeutic biological agent or a salt thereof in the composition is greater than about 250 mg / mL.

[0009] The compositions are useful, in some embodiments, for administering highly concentrated suspensions of therapeutic biologic particles via syringe injection, ambulatory drug delivery injection devices, or orally administered liquid syringe capsules without the need for manual premixing or resuspension prior to administration. [Brief description of the drawings]

[0010] [Figure 1] 1 shows an image of particles used in the compositions disclosed herein at 1000x magnification. [Diagram 2] Shown are images of the flocculation volume in the presence and absence of different flocculating agents (lecithin, PS80) at multiple concentrations. [Diagram 3] 1 shows a graph depicting the stability of IgG particles alone and in suspension during storage at 40° C. over 12 months. [Figure 4] 4 shows plots of parameters for flocculant and protein concentration. [Diagram 5] 1 shows a plot of the injectability of suspensions using caprylic triglyceride as the liquid carrier. [Figure 6] 1 shows plots of aggregate volume over time at various temperatures. [Figure 7] 1 shows a plot of the injectability of the suspension over time at various temperatures, averaging a force of about 2.4 N. [Figure 8] 1 shows a plot of injection force for various needle sizes. [Figure 9] 1 shows a plot of injection force versus injection time using a 27 gauge UTW needle. [Figure 10A] Representative images of the agglomeration volume during capsule storage over time are shown. [Figure 10B] Representative images of the agglomeration volume during capsule storage over time are shown. [Figure 11] 1 shows graphs of the pharmacokinetic profiles for mAb microparticle suspension (SC injection) and aqueous mAb SC injection in a cohort of rats (Sprague Dawley). [Figure 12] 1 shows an image of rituximab particles used in the compositions disclosed herein at a magnification of 30 μm. [Figure 13] 1 shows an image of a Rituximab particle that has been sectioned to reveal an internal cross section. [Figure 14] 1 shows a plot of the gliding force of a 620 mg / mL rituximab suspension with aggregating agent when injected. [Figure 15] 1 shows an image of rituximab particles used in the compositions disclosed herein at a magnification of 30 μm. [Figure 16] 1 shows an image of a Rituximab particle that has been sectioned to reveal an internal cross section. [Figure 17] 1 shows a plot of the gliding force of a 507 mg / mL rituximab suspension with aggregating agent when injected. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Therapeutic biologics, particularly monoclonal antibody (mAb) therapeutics, have dramatically improved the treatment of human diseases. However, the delivery of these biologics places a burden on the patient. The standard of administration is often by intravenous (IV) infusion at low concentrations, which can take hours to deliver, causing patient discomfort and increasing the risk of infection to the patient. Subcutaneous (SC) delivery by simple injection is preferred, but constraints on SC delivery volumes (1.5-2.0 mL) necessitate antibody concentrations above 100 mg / mL, which is often not feasible. Solution concentrations above 100 mg / mL are highly viscous, which leads to very high injection forces, often propagating degradation of the therapeutic antibody composition. Utilization of particle suspension technology can deliver therapeutic biologic (e.g., antibody) concentrations above 500 mg / mL while preserving the intact structure and bioactivity of the therapeutic biologic (e.g., mAb), thus providing benefits to patients, healthcare providers, payers, and pharmaceutical developers by converting the delivery of therapeutic biologics from IV to SC. However, therapeutic biologic particles will eventually settle or settle out of the suspension medium (e.g., pharma- ceutically acceptable liquid carrier) over time, leading to high injection forces. Thus, manual agitation or premixing of the container closure by external means (e.g., vortexing or sonic agitation) is required for particle resuspension prior to administration of the composition.

[0012] The present disclosure generally relates to pharma- ceutically effective compositions and methods comprising a plurality of particles suspended in a pharma- ceutically acceptable liquid carrier, the particles (e.g., substantially all of the particles) comprising at least one therapeutic biological agent or salt thereof, and a flocculating agent, wherein the concentration of the flocculating agent in the composition is less than about 50 mg / mL. In some embodiments, the concentration of the therapeutic biological agent or salt thereof in the composition is greater than about 250 mg / mL. In some embodiments, the compositions and methods described herein further comprise administering a pharma- ceutically effective amount of at least one hyaluronan degrading agent (e.g., hyaluronidase), which can be administered simultaneously, sequentially, or intermittently with the composition. In some embodiments, the plurality of particles and the flocculating agent remain substantially suspended in the non-aqueous liquid carrier for at least one month. In certain embodiments, the composition is administered by syringe injection, portable drug delivery injection device, or orally administered liquid injector capsule without the need for manual premixing or resuspension prior to administration. In some embodiments, the injection force of the composition remains substantially the same for at least one month under container-closed storage conditions at less than about 40° C. See Examples 2-3 herein for a description of an example composition of the present disclosure comprising a plurality of particles suspended in a pharma- ceutically acceptable liquid carrier with a flocculating agent.

[0013] It is easily understood that the aspects and embodiments generally described herein are exemplary. The following more detailed description of various aspects and embodiments is not intended to limit the scope of the present disclosure, but is merely representative of various aspects and embodiments. Furthermore, the compositions and methods disclosed herein may be modified by those skilled in the art without departing from the scope of the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. All publications and patents referenced herein are incorporated by reference.

[0014] definition For purposes of this disclosure, the following definitions will be used unless expressly stated otherwise.

[0015] The terms "a", "an", "the" and similar referents used in the context of describing the present disclosure should be construed to include both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. All compositions described herein can be performed in any suitable manner, unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended merely to better clarify the disclosure and does not impose limitations on the scope of the disclosure as otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0016] The term "about" in connection with given numerical values, such as temperature and time period, is meant to include numerical values ​​within 10% of the specified value.

[0017] As used herein, the term "administer" or "administered" refers to the actual physical introduction of a composition into or onto a subject (as appropriate). Any and all methods of introducing a composition into a subject are contemplated in accordance with the present disclosure, and the composition is not dependent on, and should not be construed as being dependent on, any particular means of introduction. Means of introduction are known to those of skill in the art and are exemplified herein.

[0018] As used herein, an "alkyl" group or "alkane" is a straight or branched chain non-aromatic hydrocarbon that is fully saturated. Typically, a straight or branched chain alkyl group has from 1 to about 20 carbon atoms, preferably from 1 to about 10 carbon atoms, unless otherwise defined. Examples of straight and branched chain alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, 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. 1-8Straight-chain or branched alkyl groups are also referred to as "lower alkyl" groups. Alkyl groups with two open valences are sometimes referred to as alkylene groups, such as methylene, ethylene, propylene, etc. Furthermore, the term "alkyl" (or "lower alkyl") as used throughout the specification, examples, and claims is intended to include both "unsubstituted alkyl" and "substituted alkyl," the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents, unless otherwise specified, can include, for example, alkyl, halogen, hydroxyl, carbonyl (such as carboxyl and alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties. Those skilled in the art will understand that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For example, the substituents of substituted alkyls can include substituted and unsubstituted forms of amino, azido, imino, amido, phosphoryl (including phosphonates and phosphinates), sulfonyl (including sulfates, sulfonamides, sulfamoyl, and sulfonates), and silyl groups, as well as ethers, alkylthio, carbonyl (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN, and the like. Exemplary substituted alkyls are described below. Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls, -CF3, -CN, and the like. In some embodiments, the term "alkyl" can refer to "cycloalkyl," which refers to a non-aromatic carbocyclic ring having 3 to 10 carbon ring atoms that are carbon atoms bonded together to form a ring. The ring can be saturated or can have one or more carbon-carbon double bonds.Examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, and cycloheptyl, as well as bridged 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 can be pentane, hexane, heptane, octane, cyclohexane, and the like, or combinations thereof. Aromatic hydrocarbon solvents can be benzene, toluene, and the like, or combinations thereof. Alcohols or alkyl alcohols include, for example, methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, decanol, amyl alcohol, or combinations thereof. Alkyl ethers include methyl, ethyl, propyl, butyl, and the like, for example, diethyl ether, diisopropyl ether, or combinations thereof. Sulfoxides include dimethyl sulfoxide (DMSO), decyl methyl sulfoxide, tetradecyl methyl sulfoxide, and the like, or combinations thereof. The term "alkyl ketone" refers to a ketone substituted with an alkyl group, for example, acetone, ethyl methyl ketone, and the like, or combinations thereof. The term "alkyl acetate" refers to an acetate substituted with an alkyl group, for example, 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, activated-pentyl acetate), 2-ethylhexyl acetate, and the like, or combinations thereof.The term "alkyl formate" refers to a formate substituted with an alkyl group, such as methyl formate, ethyl formate, propyl formate, butyl formate, etc., or combinations thereof. The term "trialkylamine" refers to an amino group substituted with three alkyl groups, such as triethylamine.

[0019] As used herein, "amino acid" or "residue" refers to any naturally occurring or non-naturally occurring amino acid, any amino acid derivative, or any amino acid mimetic known in the art. L- and D-forms of each amino acid are included, with the L-form usually being preferred. In some embodiments, the term refers to any one of the 20 naturally occurring amino acids: the L-form 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 can 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 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, sodium sulfate, and the like.

[0020] As used herein, unless the context otherwise requires, the term "comprise", as well as variations of the term, such as "comprising", "comprises", and "comprised", are not intended to exclude additional additives, components, elements, or steps. The terms "including" and "comprising" may be used interchangeably. As used herein, phrases such as "selected from the group consisting of", "selected from" and the like include mixtures of the specified materials. When a numerical limit or range is stated herein, the endpoints are included. Also, all values ​​and subranges within a numerical limit or range are specifically included as if expressly written out herein. Reference to an element in the singular is intended to mean "one or more" and not "one and only one" unless specifically stated otherwise. Terms such as "some" refer to one or more, and singular terms such as "a", "an", and "the" refer to one or more, unless specifically stated otherwise.

[0021] The term "oligopeptide" is used to refer to a peptide with few amino acid members, as opposed to a polypeptide or protein. 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), icosapeptides (20 amino acids), tricontapeptides (30 amino acids), tetracontapeptides (40 amino acids), and the like. Oligopeptides may also be classified according to molecular structure: aeruginosins, cyanopeptolins, microcystins, microviridins, microginins, anabaenopeptins, and cyclamides, and the like. Homo-oligopeptides are oligopeptides that contain the same amino acids. In certain embodiments, homo-oligopeptides contain polyvaline, polyalanine, and polyglycine hexamers of 10 amino acids.

[0022] The meaning of the term "peptide" is defined as a small protein of two or more amino acids linked by the carboxyl group of one amino acid to the amino group of another amino acid. Thus, 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 has about 2 to about 100 amino acids, while a polypeptide or protein has about 100 or more amino acids, up to the full length sequence that can be translated from a gene. In addition, as used herein, a peptide can be a subsequence or portion of a polypeptide or protein. In certain embodiments, 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, 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 acids in length.

[0023] As used herein, the term "pharmaceutical acceptable" refers to a composition that, within the scope of sound medical judgment, is suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response, or other problem or complication, and is commensurate with a reasonable benefit / risk ratio. Preferably, the term "pharmaceutical acceptable" means approved by a federal or state government regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, and more particularly in humans.

[0024] The meaning of the term "protein" is defined as a linear polymer built from about 20 different amino acids. The type and sequence of amino acids in a protein are specified by the DNA that produces them. In certain embodiments, the sequence can be natural and non-natural. The sequence of amino acids determines the overall structure and function of the protein. In some embodiments, a protein can contain 50 or more residues. In certain embodiments, a protein can contain a length of more than about 101 residues. The net charge of a protein can be determined by two factors: 1) the total count of acidic versus basic amino acids, and 2) the particular solvent pH environment that exposes the positive or negative residues. As used herein, a "net positively or net negatively charged protein" is a protein that has a net positive or net negative charge under a non-denaturing pH environment. In general, one of skill in the art will recognize that all proteins can be considered "net negatively charged proteins" regardless of their amino acid composition, 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. Proteins may function as structural materials (e.g., keratin), enzymes, hormones, transporters (e.g., hemoglobin), antibodies, or regulators 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 biologic. In certain embodiments, the protein is bovine serum albumin (BSA) or human serum albumin (HSA).

[0025] As used herein, the term "substantially" refers to a majority or majority, 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.

[0026] It is understood that the particular order or hierarchy of steps in the disclosed processes is illustrative of example approaches. It is understood that the particular order or hierarchy of steps in the processes may be rearranged based on design preferences. Some steps may be performed simultaneously. A phrase such as "embodiment" does not imply that such embodiment applies to all configurations of the subject technology. Disclosure of an embodiment may apply to all embodiments, or to one or more embodiments. A phrase such as embodiment may refer to one or more embodiments, or vice versa.

[0027] particle Unless otherwise defined, all terms, notations, and other scientific terms used herein are intended to have the meaning commonly understood by those skilled in the art to which this disclosure belongs. In some cases, terms with commonly 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 that commonly 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 methodology. Where appropriate, unless otherwise specified, procedures involving the use of commercially available kits and reagents are generally performed according to manufacturer-defined protocols and / or parameters. As used herein, the term "and / or," when used in a list of two or more items, means that any one of the listed items can be used alone, or any combination of two or more of the listed items can 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 only, B only, C only, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination.

[0028] The terms "particle" or "particles" or "microparticle" or "microparticles" as used herein interchangeably refer to discrete bodies or bodies in the broadest sense. The particles described herein are circular and of controlled dispersity with characteristic sizes ranging from submicrometers to tens of micrometers, as opposed to the porous monolithic "cakes" typically produced during conventional freeze-drying, for example. This morphology allows for flowable powders (e.g., characterized by low Hausner ratios) without post-processing. In some embodiments, the term "particle" refers to a quantity of protein or proteins, e.g., therapeutic biologic or therapeutic biologics, in any state of matter that is substantially solid compared to a liquid droplet.

[0029] As disclosed herein, a therapeutic biological product, also known as a biologic pharmaceutical product or biopharmaceutical, is any pharmaceutical drug produced 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 therapy, tissues, and recombinant therapeutic proteins. Biological products can be isolated from a variety of natural sources, e.g., human, animal, or microorganism, and can be produced by biotechnological methods or other techniques. In some embodiments of the present 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 an antibody fragment. In some embodiments, the compositions described herein comprise at least two plurality of particles (e.g., a first and a second plurality of particles) suspended in a pharma- ceutically acceptable liquid carrier, each plurality of particles comprising at least one therapeutic biologic (e.g., at least one different therapeutic biologic compared to the particles in the other plurality). In some embodiments, the compositions described herein comprise at least two plurality of particles (e.g., a first and a second plurality of particles) suspended in a pharma-ceutically acceptable liquid carrier, each plurality of particles comprising at least one antibody, e.g., a plurality of rituximab particles and a plurality of trastuzumab particles.

[0030] The terms "antibody" and "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal, polyclonal, multivalent, and multispecific antibodies, regardless of how they are produced, i.e., using immunization, recombinant, or synthetic methodologies. Antibodies may 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 bodies. Antibodies can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated alpha, delta, epsilon, gamma, and mu, respectively. The gamma class is further divided into subclasses based on differences in sequence and function, for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In some embodiments of the present disclosure, the IgG antibody is an IgG1 antibody. In certain embodiments of the present disclosure, the IgG antibody is a monoclonal IgG antibody. The light chain from any vertebrate species can be assigned to one of two clearly distinct types, e.g., kappa and lambda, based on the amino acid sequence of their constant domain.

[0031] Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. In some embodiments, light chains are classified as either kappa or lambda. In some embodiments, heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. In certain embodiments of the present disclosure, the antibody is an IgG antibody.

[0032] An exemplary antibody (immunoglobulin) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The terms "variable light" chain, domain, region, and component are used interchangeably and are referred to as "VL" or "VL". L " and refers to the light chain of an antibody or antibody fragment. Similarly, the terms "variable heavy" chain, domains, regions, and components are used interchangeably and are abbreviated as "VH" or "V H " and 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 a 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. H and L chains define specific Ig domains. In particular, each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the alpha and gamma chains, and four CH domains for the p and c isotypes. Each L chain has a variable domain (VL) at the N-terminus, followed by a constant domain (CL) at its other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain (CHL) of the heavy chain. The constant domain comprises the Fc portion, which comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of the antibody, such as ADCC, are determined by sequences within the Fc region, which is also the portion recognized by Fc receptors (FcRs) found on certain types of cells.

[0033] As disclosed herein, the pairing of VH and VL together forms a "variable region" or "variable domain" comprising the amino-terminal domain of the heavy or light chain of an 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 the "antigen binding site", which influences antigen binding and defines the specificity of a particular antibody for its particular antigen. The V region spans approximately 110 amino acid residues and consists of relatively invariant stretches called framework regions (FRs) (generally about 4) of 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions" (generally about 3), each generally 9-12 amino acids long. The FRs largely adopt a p-sheet configuration, and the hypervariable regions form loops that connect, and in some cases form part of, the p-sheet structure. In certain embodiments, "hypervariable region" refers to the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies contain six hypervariable regions, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0034] The terms "full length antibody", "intact antibody" and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form, and not as an antibody fragment as defined above. The term particularly refers to an antibody having a heavy chain containing an Fc region. A full length antibody may be a native sequence antibody or an antibody variant. In certain embodiments, an "intact" or "whole" antibody is one that comprises an antigen binding site, as well as a CL and at least the heavy chain constant domains, CH1, CH2, and CH3. The constant domain may be a native sequence constant domain, e.g., a human native sequence constant domain, or an amino acid sequence variant thereof.

[0035] As noted above, the term antibody as used herein includes, unless otherwise indicated or clearly contradicted by context, fragments of antibodies that retain the ability to specifically interact with, e.g., bind to, an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antibody" include (i) Fab' or Fab fragments, monovalent fragments consisting of a light chain variable domain (VL), a heavy chain variable domain (VH), a light chain constant region (CL), and a heavy chain constant region domain 1 (CH1) or monovalent antibodies as described in WO2007 / 059782; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting essentially of the VH and CH1 domains; (iv) Fv fragments consisting essentially of the VL and VH domains of a single arm of an antibody; (v) Holt et al; Trends Biotechnol. 2003 November; 21(11):484-90, also called domain antibodies; (vi) camelids or nanobodies Revets et al. al; Expert Opin Biol Ther. 2005 January; 5(1): 111-24, and (vii) 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 recombinant methods by a synthetic linker that allows them to be produced as a single protein chain in which the VL and VH domains pair to form a monovalent molecule (known as single chain antibodies or single chain Fvs (scFvs), 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 encompassed within the scope of the term antibody unless otherwise specified or clearly dictated by the context.Although such fragments are generally included within the meaning of antibody, they are taken together and each independently is a unique feature of the present invention and exhibits different biological properties and utility. These and other useful antibody fragments in the context of the present invention are discussed further herein.

[0036] As disclosed herein, "whole antibody fragments containing variable domains" 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 together with the variable region domain (VH) of the H chain and the first constant domain (CHI) of one heavy chain. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. A "Fab' fragment" differs from a Fab fragment by having a few additional residues at the carboxy terminus of the CHI domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for a Fab' in which the cysteine ​​residue(s) of the constant domain bear a free thiol group. A "F(ab')2 fragment" roughly corresponds to two disulfide-linked Fab fragments with bivalent antigen-binding activity and is still capable of cross-linking to antigen. An "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of one heavy chain variable region domain and one light chain variable region domain in tight non-covalent association. In single chain Fv (scFv) species, one heavy chain variable domain and one light chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure similar to that of two chain Fv species. From the folding of these two domains arise six hypervariable loops (three loops each 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 comprising VH and VL antibody domains connected to form a single polypeptide chain. In certain embodiments, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. In some embodiments, a "single variable domain" is half of an Fv (containing only the three CDRs specific for an antigen) that has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0037] In some embodiments, "diabody" refers to an antibody fragment with two antigen binding sites, which comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). Small antibody fragments are prepared by constructing sFv fragments with a short linker (about 5-10 residues) between the VH and VL domains such that interchain pairing rather than intrachain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., a fragment with two antigen binding sites. In some embodiments, diabodies may be bivalent or bispecific. In certain embodiments, bispecific diabodies are heterodimers of two "crossover" sFv fragments, in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Triabodies and tetrabodies are also commonly known in the art.

[0038] An "antigen-binding fragment" of an antibody as described herein comprises only a portion of an intact antibody, and generally comprises the antigen-binding site of the intact antibody, and thus retains the ability to bind to an antigen. Illustrative examples of antibody fragments encompassed by this definition include, but are not limited to, (i) a Fab fragment having the VL, CL, VH, and CH1 domains, (ii) a Fab' fragment, which is a Fab fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain, (iii) an Fd fragment having the VH and CH1 domains, (iv) an Fd' fragment having the VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain, (v) an Fv fragment having the VL and VH domains of a single arm of an antibody, (vi) a d fragment consisting of a VH domain, and (vii) a d fragment consisting of a VH domain. Ab fragments, (vii) isolated CDR regions, (viii) F(ab')2 fragments, bivalent fragments comprising two Fab' fragments linked by a disulfide bridge at the hinge region, (ix) single chain antibody molecules, e.g., single chain Fv, scFv, (x) "diabodies" 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, (xi) "linear antibodies" comprising a pair of tandem Fd, segments (VH-CH1-VH-CH1) that together with complementary light chain polypeptides form a pair of antigen binding regions. In some embodiments, "antigen binding site" generally refers to a molecule that comprises at least the hypervariable and framework regions 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 a dAb, Fab, Fd, Fv, F(ab')2, or scFv) in the methods described herein. In some embodiments, an antigen-binding fragment competes for antigen binding with an intact antibody, eg, the intact antibody from which the fragment is derived.

[0039] 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 antibodies, 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 combination, assembly, or conjugation of such antigen-binding fragments.

[0040] In some embodiments, the term "single-chain Fv" or "scFv" or "single-chain" antibody can refer to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of sFvs, see Pluckthun, THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

[0041] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies (mAbs) are highly specific, directed against a single antigenic site or determinant on an antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. Monoclonal antibodies may be prepared by hybridoma methodology. Monoclonal antibodies may also be isolated from phage antibody libraries using molecular engineering techniques. The monoclonal antibodies of the present 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 of a single species in which all antibody molecules recognize the same epitope, since all antibody-producing cells are derived from a single B-lymphocyte cell line. Methods for producing monoclonal antibodies (mAbs) generally begin along the same lines as methods for preparing polyclonal antibodies. In some embodiments, rodents such as mice and rats are used for the production of monoclonal antibodies. In certain embodiments, rabbit, sheep, or frog cells are used for the production of 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 fusions. In some embodiments of the present disclosure, the antibody is a monoclonal antibody. In certain embodiments of the present disclosure, the IgG antibody is monoclonal.

[0042] In some embodiments, recombinant antibody fragments can be isolated from phage antibody libraries using techniques well known in the art. See, e.g., Clackson et al., 1991, Nature 352:624-628; Marks et al., 1991, J. Mol. Biol. 222:581-597. Recombinant antibody fragments can be derived from large phage antibody libraries produced by recombination in bacteria (Sblattero and Bradbury, 2000, Nature Biotechnology 18:75-80, and as described herein). Polynucleotides encoding the VH and VL components of antibody fragments, i.e., scFvs, can be used to generate recombinant full-length immunoglobulins using methods known in the art (see, e.g., Persic et al., 1997, Gene 187:9-18).

[0043] An "isolated antibody" is one that has been identified and separated and / or recovered from components of its existing environment. Contaminating components are materials that would interfere with therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.

[0044] As used herein, "human antibody" refers to an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Human antibodies can be prepared by administering antigen to transgenic animals that have been modified to produce such antibodies in response to an antigenic challenge, but whose endogenous loci have been disabled. "Humanized" forms of non-human, e.g., rodent, antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a 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 capacity. In some embodiments, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may include residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody will optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0045] An "affinity matured" antibody is one that has one or more alterations in one or more hypervariable regions thereof that result in an improvement in the affinity of the antibody for an antigen compared to a parent antibody that does not possess those alterations. In some embodiments, an affinity matured antibody can have micromolar affinity for a target antigen. In some embodiments, an affinity matured antibody can have nanomolar or even picomolar affinity for a target antigen. Affinity matured antibodies are produced by procedures known in the art.

[0046] A "blocking" or "antagonist" antibody is one that inhibits or reduces the biological activity of the antigen to which it binds. In some embodiments, a blocking 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 a polypeptide of interest. As used herein, the terms "inhibit," "inhibit," "inhibiting," and the like, in relation to the interaction of a protein inhibitor, e.g., an antagonist, refer to adversely affecting, e.g., reducing, the activity or function of a protein relative to the activity or function of the protein in the absence of the inhibitor. In some embodiments, inhibition refers to a reduction in a disease or a symptom of a disease. In some embodiments, inhibition refers to a reduction in the activity of a signal transduction pathway or a signaling pathway. Thus, inhibition includes, at least in part, partially or completely blocking a stimulus, reducing, preventing, or delaying activation, or inactivating, desensitizing, or downregulating a signal transduction or enzyme activity or the amount of a protein.

[0047] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule, e.g., an antibody, and its binding partner, e.g., an antigen. Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity, reflecting a 1:1 interaction between a member of a binding pair, e.g., an antibody and an antigen. The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, while high affinity antibodies generally bind antigens faster and tend to remain bound longer. Various methods of measuring binding affinity are known in the art, any of which can be used for the purposes of this disclosure. "Epitope" generally refers to that portion of an antigen that is bound by an antigen-binding site of an antibody. In some embodiments, an epitope may be "linear" in the sense that the hypervariable loops of the antibody CDRs that form the antigen binding site bind to a sequence of amino acids as they are in the primary protein structure. In some embodiments, an epitope is a "conformational epitope", i.e., the hypervariable loops of the CDRs bind to residues as they are presented in the tertiary or quaternary protein structure.

[0048] In some embodiments of the aforementioned compositions, the protein, e.g., therapeutic biologic, is an antibody. In some embodiments, the antibody may include, but is not limited to, 3F8, abagovomab, abatacept, abciximab, avituzumab, abrezekimab, abrilumab, acritumomab, actoxumab, avituzumab, adalimumab-adbm, adalimumab-atto, adalimumab-bwwb, adecatumumab, Ado-trastuzumab emtansine, aducanumab, afacevicumab, afelimomab, aflibercept, afutuzumab, alacizumab pegol, ALD518, alefacept, alemtuzumab, aliloc Mab, altumomab pentetate, amatuximab, anatumomab mafenatox, andecaliximab, anetumab vegetansine, anifrolumab, anrukinzumab, ansuvimab, apolizumab, aprtumab ixadotin, arcitumomab, asclinbacumab, acelizumab, atezolizumab, atidolutoxumab, atinumab, atlizumab, atortivimab, atorlimumab, avelumab, azintuxizumab vedotin, bapineuzumab, basiliximab, bavituximab, BCD-100, bectumomab, bege romab, belantamab mafodotin, belatacept, belimumab, bemarituzumab, benralizumab, bermekimab, bersanlimab, bertilimumab, besilesomab, bevacizumab, bevacizumab-awwb, bezlotoxumab, biciromab, bimagrumab, bimekizumab, virutamimab, bivatuzumab mertansine, bleselumab, blinatumomab, brontuzumab, brosozumab, bococizumab, brazikumab, brentuximab vedotin, briakinumab, brodalumab, brolucizumab, broticutuzumab, bros Mab, cabilalizumab, camidarumab tesirin, camrelizumab, canakinumab, cantuzumab mertansine, cantuzumab lavtansine, caplacizumab, capromab pendetide, carlumab, carotuximab, catumaxomab, cBR96-doxorubicin immunoconjugate, cedelizumab, cemiplimab, sergituzumab amnaleukin, sergituzumab amnaleukin, certolizumab pegol, cetrelimab, cetuximab, civisatamab, cimutuzumab, sitatuzumab bogatox, cixutumumab, clazakizumab,Clenoliximab, clivatuzumab tetraxetan, codrituzumab, cofetuzumab peridotin, cortuximab lavtansine, conatumumab, concizumab, cosfrobiximab, crenezumab, CR6261, crizanlizumab, clotezumab, cusatuzumab, dacetuzumab, daclizumab, darotuzumab, dapirorizumab pegol, daratumumab, dectrekumab, demcizumab, denileukin diftitox, denintuzumab mafodotin, denosumab, depatuxizumab mafodotin, delrotuximab biotin, detumomab, desamizumab, diazepam, Nutuximab, Ziridabuumab, Domagrozumab, Dorlimomab Alitox, Dostarlimab, Drozitumab, DS-8201, Durigotumab, Dupilumab, Durvalumab, Dusigitumab, Duvortuxizumab, Eclomeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Eldelumab, Elezanumab, Ergemtumab, Elotuzumab, Ercilimomab, Emactuzumab, Emibetuzumab, Emicizumab, Enapotamab vedotin, Enavatuzumab, Enfortumab vedotin, Enlimomab pegol, Enobu Rituzumab, Enokizumab, Enotikumab, Ensituximab, Epitumomab-situxetan, Epoetin-alfa, Epoetin-alfa-epbx, Epratuzumab, Eptinezumab, Erenumab, Erlizumab, Ertumaxomab, Etanercept, Etanercept-szzs, Etaracizumab, Etigilimab, Etrolizumab, Ebinacumab, Evolocumab, Exibirumab, Factor VIII Fc fusion protein, Factor IX Fc fusion protein, Fanolesomab, Faralimomab, Faricimab, Farletuzumab, Fasinumab, Farbi Zumab, Fezakinumab, Fivatuzumab, Ficlatuzumab, Figitumumab, Filgrastim, Filgrastim-sndz, Filibutomab, Flavourumab, Fretikumab, Flotetuzumab, Hontolizumab, Foramumab, Folavirumab, Fremanezumab, Fresolimumab, Flobocimab, Furnevetomab, Furlanumab, Futuximab, Galcanezumab, Galiximab, Ganitumab, Gantenerumab, Gatipotuzumab, Gavirimomab, Gedibumab, Gemtuzumab Ozogamicin, Gevokizumab, Gilvetomab, Gimsilumab, Girentuximab,Glenbatumumab vedotin, golimumab, gomiliximab, goslanemab, guselkumab, ibalizumab, IBI308, ibritumomab tiuxetan, icrucumab, idarucizumab, ifavotuzumab, igovomab, iradatuzumab vedotin, IMAB362, imalumab, imaprelimab, imciromab, imgatuzumab, inlacumab, indatuximab vedotin, inebilizumab, infliximab, infliximab-abda, infliximab-dyyb, infliximab-qbtx, intestinal , inolimomab, inotuzumab ozogamicin, ipilimumab, iomab-B, iratumumab, isatuximab, iscalimab, istiratumab, itolizumab, ixekizumab, keliximab, labetuzumab, lacunotuzumab, radiratuzumab vedotin, lambrolizumab, lampalizumab, lanadelumab, landgrozumab, laprituximab emtansine, ralcaviximab, lebrikizumab, lemaresomab, lendalizumab, lembervimab, lenzilumab, lerdelimumab, leronlimab, lesofabumab, letolizumab, lexatumumab, Ribivirumab, Rifastuzumab vedotin, Ligelizumab, Loncastuximab tesirin, Rosatuximab vedotin, Rilotumab satetraxetan, Lintuzumab, Lirilumab, Roderucizumab, Loxivetomab, Lorvotuzumab mertansine, Lucatumumab, Lulizumab pegol, Rumiriximab, Lumuletuzumab, Rupartumab amadotin, Rutikizumab, Maftivimab, Mapatumumab, Margetuximab, Marstacimab, Maslimomab, Mavrilimumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mikal Mab, mirvetuximab soravtansine, mitumomab, modotuximab, mogamulizumab, monalizumab, morolimumab, mosunetuzumab, motavizumab, moxetumomab pasudotox, muromonab-CD3, nacolomab butafenatox, namilumab, naptumomab estafenatox, naratuximab emtansine, narutumab, natalizumab, navicixizumab, navicibumab, naxitamab, nebacumab, necitumumab, nemolizumab, NEOD001, nerelimomab, nesbacumab, netakimab, nimotuzumab, nirsevimab, nivolumab,nofetumomab merpentan, obiltoxaximab, obinutuzumab, ocaratuzumab, ocrelizumab, odesivimab, odesivimab-ebgn, ozlimomab, ofatumumab, olaratumab, oleculab, orendalizumab, olokizumab, omalizumab, omburtamab, OMS721, onartuzumab, ontuxizumab, ombatilimab, opicinumab, oportuzumab monatox, oregovomab, olticumab, otelixizumab, otilimab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, pa Rivizumab, Pamrevlumab, Panitumumab, Pancomab, Panobacumab, Palsatuzumab, Pascolizumab, Pasotuxizumab, Pateclizumab, Patritumab, PDR001, Pegfilgrastim-jmdb, Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placlumab, Prozalizumab, Pogalizumab, Polatuzumab vedotin, Ponezumab, Polgabiximab, Prasinezumab, Prezarizumab, Priliximab, Pritoxaximab, Pritumumab, P RO140, Kirizumab, Racotumomab, Ladolezumab, Rafivirumab, Ralpancizumab, Ramucirumab, Ranevetomab, Ranibizumab, Raxibacumab, Ravagalimab, Ravulizumab, Refanezumab, Regavirumab, Relatolimab, Lemtolumab, Reslizumab, Rilonacept, Rilotumumab, Linucumab, Risankizumab, Rituximab, Rituximab-abbs, Rituximab-arrx, Rituximab-pvvr, Ribazumab pegol, Ribazumab pegol, Lobatumumab, Rmab, Lorezumab, Romilkimab, Romiplostim, Romo Sozumab, Rontalizumab, Rosmantuzumab, Robalpituzumab Tecilline, Robalpituzumab Tecilline, Rovelizumab, Rozanolixizumab, Ruplizumab, Sacituzumab govitecan, Samalizumab, Samrotamab vedotin, Sapelizumab, Sarilumab, Satralizumab (SA237), Satumomab pendetide, Secukinumab, Celiclerumab, Seribantumab, Setoxaximab, Setrusumab, Sevirumab, Sibrotuzumab, SGN-CD19A, SGN-CD33A, SHP647, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab,Siltratumab vedotin, sirukumab, sofituzumab vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, spartalizumab, stamulumab, sulesomab, sputabumab, stimulimab, subizumab, sublatokisumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, tafasitamab, talicozumab, talizumab, tamtubetomab, tanezumab, tapritumomab paptokis, tarexizumab, taborimab, tefibazumab, terimomab aritokis s, telisotuzumab vedotin, tenatumomab, teneliximab, teplizumab, tepositamab, teprotumumab, tesidolumab, tetulomab, tezepelumab, TGN1412, tiburizumab, ticilimumab, tildrakizumab, tigatuzumab, timigtuzumab, timolumab, tiragotumab, tislelizumab, tisotuzumab vedotin, TNX-650, tocilizumab, tonzotuximab, toralizumab, tosatoxumab, tositumomab, tobetumab, tralokinumab, trastuzumab, trastuzumab-anns, trastuzumab-dkst, trastuzumab-dttb, trastuzumab emtansine, trastuzumab-pkrb, tregalizumab, tremelimumab, trevogrumab, tucotuzumab celmoleukin, tuvilumab, ublituximab, urocupulumab, urelumab, urtoxazumab, ustekinumab, utomilumab, vadastuximab butarilin, banalimab, bundletuzumab vedotin, vanticizumab, vanucizumab, bapaliximab, valisac Mab, varlilumab, batelizumab, vedolizumab, veltuzumab, beparimomab, besenkumab, visilizumab, bovalilizumab, volociximab, bonlerolizumab, bopratelimab, borsetuzumab mafodotin, votumumab, xentuzumab, XMAB-5574, zalutumumab, zanolimumab, zatuximab, zenoctuzumab, diralimumab, zolbetuximab (IMAB362, claudiximab), Ziv-aflibercept, or zolimomab alitoxin.

[0049] In some embodiments of the above-mentioned composition, the antibody is monoclonal. In certain embodiments, the monoclonal antibody includes, but is not limited to, 3F8, 8H9, abatacept, abagovomab, abciximab, avituzumab, adalimumab-adbm, adalimumab-atto, adalimumab-bwwb, abrilumab, actoxumab, avituzumab, abrezekimab, abrilumab, actoxumab, adalimumab, adecatumumab, Ado-trastuzumab emtansine, aducanumab, afacevicumab, afelimomab, aflibercept, afutuzumab, alacizumab pegol, ALD518, , alefacept, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, anatumomab mafenatox, andecaliximab, anetumab ravtansine, anifrolumab, anrukinzumab (IMA-638), ansuvimab, apolizumab, arcitumomab, asclinbacumab, acelizumab, atezolizumab, atidortoxumab, atinumab, atlizumab (tocilizumab), atortivimab, atorlimumab, avelumab, bapineuzumab, basiliximab, bevacizumab, bevacizumab-awwb , BCD-100, bectumomab, begelomab, belatacept, belimumab, bemarituzumab, benralizumab, bermekimab, bersanlimab, bertilimumab, besilesomab, bezlotoxumab, biciromab, bimagrumab, bimekizumab, virutamimab, bivatuzumab mertansine, bleselumab, blinatumomab, brontuzumab, brosozumab, bococizumab, brazikumab, brentuximab vedotin, briakinumab, brodalumab, brolucizumab, broticutuzumab, burosumab, cabilalizumab, camrelizumab canakinumab, cantuzumab mertansine, cantuzumab lavtansine, caplacizumab, capromab pendetide, carlumab, carotuximab, catumaxomab, cedelizumab, cemiplimab, certolizumab pegol, cetrelimab, cetuximab, civisatamab, cimutuzumab, Ch.14.18, sitatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, codrituzumab, cofetuzumab peridotin, coltuximab lavtansine, conatumumab, concizumab,Cosfrobiximab, crenezumab, CR6261, crizanlizumab, clotezumab, cusatuzumab, dacetuzumab, daclizumab, darotuzumab, dapirorizumab pegol, daratumumab, dectrekumab, demcizumab, denileukin diftitox, denintuzumab mafodotin, denosumab, delrotuximab biotin, detumomab, desamizumab, dinutuximab, ziridabumab, domagurozumab, dorlimomab alitox, dostarlimab, drozitumab, durigotumab, dupilumab, durvalumab, dusigitumab, duvortuximab zumab, ecromeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab, eldelumab, elezanumab, elgemtumab, elotuzumab, ersilimomab, emactuzumab, emibetuzumab, emicizumab, enabatuzumab, enfortumab vedotin, enlimomab pegol, enoblitzumab, enokizumab, enoticumab, ensituximab, epitumomab situxetan, epoetin-alpha, epoetin-alpha-epbx, epratuzumab, eptinezumab, erenumab, erlizumab, ertumaxomab , etanercept, etanercept-szzs, etaracizumab, etigilimab, etrolizumab, evinacumab, evolocumab, exibirumab, factor VIII Fc fusion protein, factor IX Fc fusion protein, fanolesomab, faralimomab, faricimab, farletuzumab, fasinumab, FBTA05, felvizumab, fezakinumab, fibatuzumab, ficlatuzumab, figitumumab, filgrastim, filgrastim-sndz, filibumab, framvotumab, fretikumab, flotetuzumab, hontolizumab, hontolizumab Ralumab, folavirumab, fremanezumab, fresolimumab, furobocimab, furunevetomab, furanumab, futuximab, galcanezumab, galiximab, ganitumab, gantenerumab, gatipotuzumab, gavirimomab, gedivumab, gemtuzumab ozogamicin, gevokizumab, gilvetomab, gimsilumab, girentuximab, glenbatumumab vedotin, golimumab, gomilikimab, goslanemab, guselkumab, ibalizumab, IBI308, ibritumomab tiuxetan, icrucumab, idarucizumab, ifavotuzumab, igovomab,IMAB362, Imalumab, Imaprelimab, Imciromab, Imgatuzumab, Inclacumab, Indatuximab ravtansine, Insatumab vedotin, Inebilizumab, Infliximab, Infliximab-abda, Infliximab-dyyb, Infliximab-qbtx, Intetumumab, Inolimomab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Isatuximab, Iscalimab, Istiratumab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lacnotuzumab, Lambrolizumab, Lampalizumab , lanadelumab, landgrozumab, ralcaviximab, lebrikizumab, remaresomab, lendalizumab, lembervimab, lenzilumab, lerdelimumab, leronlimab, lesofabumab, letolizumab, lexatumumab, ribivirumab, rifastuzumab vedotin, ligelizumab, rilotumab satetraxetan, lintuzumab, lirilumab, roderucizumab, lokivetomab, lorvotuzumab mertansine, lucatumumab, lurizumab pegol, rumiliximab, lumuletuzumab, rutikizumab, maftivimab, mapatumumab, margetuximab, ma Rustacimab, Maslimomab, Mavrilimumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mirikizumab, Mirvetuximab Soravtansine, Mitumomab, Modotuximab, Mogamulizumab, Monalizumab, Morolimumab, Mosunetuzumab, Motavizumab, Moxetumomab Pasudotox, Muromonab-CD3, Nacolomab Butafenatox, Namilumab, Naptumomab Estafenatox, Narutazumab, Natalizumab, Nabicixizumab, Nabivumab, Naxitamab, Nebacumab, Necitumumab, Nemolizumab, NE OD001, nerelimomab, nesbacumab, netakimab, nimotuzumab, nirsevimab, nivolumab, nofetumomab merpentan, obiltoxaximab, obinutuzumab, ocralatuzumab, ocrelizumab, odesivimab, odesivimab-ebgn, ozlimomab, ofatumumab, olaratumab, oleculab, orendalizumab, olokizumab, omalizumab, omburtamab, OMS721, onartuzumab, ontuxizumab, ombatilimab, opicinumab, oportuzumab monatox, oregovomab, olticumab, otelixizumab,Otilimab, Otlertuzumab, Oxelumab, Ozanezumab, Pagibaximab, Palivizumab, Pamrevlumab, Panitumumab, Pancomab, Panobacumab, Palsatuzumab, Pascolizumab, Pasotuxizumab, Pateclizumab, Patritumab, PDR001, Pegfilgrastim-jmdb, Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placlumab, Prosalizumab, Pogalizumab, Polatuzumab vedotin, Ponezumab, Polgabiximab, Placid Nezumab, Prezarizumab, Priliximab, Pritoxaximab, Pritumumab, PRO140, Kirizumab, Tetulomab, Racotumomab, Ladolezumab, Rafivirumab, Ralpancizumab, Ramucirumab, Ranevetomab, Ranibizumab, Raxibacumab, Ravagalimab, Ravulizumab, Refanezumab, Regavirumab, Relatolimab, Lemtolumab, Reslizumab, Rilonacept, Rilotumumab, Linucumab, Risankizumab-Rzaa, Rituximab, Rituximab-Abbs, Rituximab-Arrx, Rituximab-Pvvr, Lobatumumab , Rmab, lorezumab, romilukimab, romiplostim, romosozumab, rontalizumab, rosmantuzumab, rovelizumab, rozanolixizumab, ruplizumab, sacituzumab govitecan, samalizumab, sarilumab, satralizumab (SA237), satumomab pendetide, secukinumab, seliclerumab, seribantumab, setoxaximab, setrusumab, sevirumab, sibrotuzumab, SGN-CD19A, SGN-CD33A, SHP647, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, sofituzumab Vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, spartalizumab, stamulumab, sulesomab, sputabumab, stimulimab, suvizumab, sublatoxumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, tafasitamab, talacuzumab, talizumab, tamtubetomab, tanezumab, taplitumomab paptokis, tarexizumab, taborimab, tefibazumab, terimomab alitokis, tenatumomab, teneliximab, teplizumab, tepositamab, teprotumumab, tesidolumab, tetulomab (rilotomab),Tezepelumab, TGN1412, tiburizumab, ticilimumab (tremelimumab), tildrakizumab, tigatuzumab, timigtuzumab, timolumab, tiragotumab, tislelizumab, TNX-650, tocilizumab (atlizumab), tomzotuximab, toralizumab, tosatoxumab, tositumomab, tobetumab, tralokinumab, Trastuzumab, trastuzumab-anns, trastuzumab-dkst, trastuzumab-dttb, trastuzumab emtansine, trastuzumab-pkrb, TRBS07, tregalizumab, tremelimumab, trevoglumab, tucotuzumab celmoleukin, tuvilumab, ublituximab, urocupulumab, urelumab, urtoxin and sazumab, ustekinumab, utomirumab, banalimab, bundletuzumab vedotin, vanticutumab, vanucizumab, bapaliximab, valisacumab, varlilumab, batelizumab, vedolizumab, veltuzumab, beparimomab, besencumab, visilizumab, bovalilizumab, volociximab, bonlerolizumab, bopratelimab, borsetuzumab mafodotin, votumumab, xentuzumab, XMAB-5574, zalutumumab, zanolimumab, zatuximab, zenoctuzumab, diralimumab, zolbetuximab (IMAB362, claudiximab), Ziv-aflibercept, zolimomab alitkis, or the corresponding anti-drug antibodies in a sample from a human patient. In certain embodiments, the monoclonal antibody is rituximab, rituximab-abbs, rituximab-arrx, or rituximab-pvvr. In some embodiments, the monoclonal antibody is atortivimab, maftivimab, odesivimab-ebgn, or a combination thereof.

[0050] In some embodiments, the monoclonal antibody is a biosimilar, including, but not limited to, adalimumab-adbm, adalimumab-atto, adalimumab-bwwb, bevacizumab-awwb, epoetin alfa-epbx, etanercept-szzs, infliximab-abda, infliximab-dyyb, infliximab-qbtx, filgrastim-sndz, odesivimab-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, follitropin 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.

[0051] In some embodiments, the targeting moiety 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 an antigenic determinant portion of an antibody, or other modified immunoglobulin molecule that contains an antigen recognition site.

[0052] In certain embodiments, the therapeutic biologic is ledipasvir / sovosbuvir, insulin glargine, lenalidomide, adsorbed 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.

[0053] In some embodiments, the therapeutic biologic is abatacept, abobotulinumtoxinA, agalsidase beta, albiglutide, aldesleukin, alglucosidase alfa, alteplase (cathflo activase), anakinra, asfotase alfa, asparaginase, asparaginase erwinia chrysanthemi, becaplermin, belatacept, collagenase, collagenase clostridium histolyticum, darbepoetin alfa, denileukin diftitox, dornase alfa, dulaglutide, ecallantide, efgaltigimod alfa-fcab, elosulfase alfa, etanercept-szzs, filgrastim, filgrastim-sndz, galsulfase, glucarpidase, idursulfase, incobotulinumtoxinA, interferon alfa-2b, interferon alfa-n3, interferon beta-1a, interferon beta-1b, interferon gamma-1b, laronidase, methoxypolyethylene glycol-epoetiamide botulinum toxin B, romiplostim, sargramostim, sebelipase alfa, tbo-filgrastim, tenecteplase, or ziv-aflibercept.

[0054] Proteins, e.g., therapeutic biologicals in particles disclosed herein, have an activity per unit of about 0.5 to about 1.0, an activity per unit of about 0.75 to about 1.0, or an activity per unit of about 0.9 to about 1.0. Activity is measured relative to the same protein prior to particle formation. In some embodiments, the protein has an activity per unit of about 0.5 to about 1.0. The term "activity" refers to the ratio of a functional or structural aspect of a protein, e.g., an antibody, an 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 feed solution immediately prior to 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, e.g., immediately after particle formation.

[0055] In some embodiments, substantially all of the particles have less than about 10% aggregation of proteins, e.g., less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the therapeutic biological after processing. In some embodiments, substantially all of the particles have less than about 5% aggregation of the therapeutic biological. In certain embodiments, substantially all of the particles have less than about 3% aggregation of the therapeutic biological. In some embodiments, substantially all of the particles have less than about 1% aggregation of the therapeutic biological. In certain embodiments, substantially all of the particles are substantially free of any aggregation of the therapeutic biological. In some embodiments, substantially all of the particles have less than about 0.5% aggregation of the therapeutic biological.

[0056] In some embodiments, substantially all of the particles have less than about 10% fragmentation of the therapeutic biological after processing, such as protein. In some embodiments, substantially all of the particles have less than about 3% fragmentation of the therapeutic biological. In certain embodiments, substantially all of the particles have less than about 1% fragmentation of the therapeutic biological. In some embodiments, substantially all of the particles are substantially free of any fragmentation of the therapeutic biological. In some embodiments, substantially all of the particles have less than about 5% fragmentation of the therapeutic biological. A suitable method for measuring aggregation and fragmentation of the therapeutic biological can be achieved by using size exclusion chromatography (SEC).

[0057] In certain embodiments, the composition provides less than about 5% (e.g., less than about 4, 3, 2, or 1%) change in charge variants in a population of proteins, e.g., therapeutic biologics, compared to the therapeutic biologics before particle formation. In some embodiments, the particles have less than about 5% change in charge variants in a population of therapeutic biologics after processing. Charge variants can be acidic, basic, or neutral, and the variation can 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 attachment of the amine moieties of two lysine residues with a reducing sugar, or conversion of an N-terminal amine to a neutral amide by cyclization of an N-terminal glutamine. Negative charges on proteins, e.g., antibodies or fragments, can appear due to conversion of asparagine residues to aspartic acid and / or isoaspartic acid residues via deamidation reactions.

[0058] In some embodiments, substantially all of the particles have less than about 5% change in charge variants of the protein, e.g., therapeutic biologic, compared to an aqueous composition containing at least one therapeutic biologic in soluble (monomeric) form prior to particle formation. In some embodiments, substantially all of the particles have less than about 3% change in charge variants of the therapeutic biologic. In certain embodiments, substantially all of the particles have less than about 1% change in charge variants of the therapeutic biologic. In some embodiments, substantially all of the particles are substantially free of any change in charge variants of the therapeutic biologic. Exemplary methods for measuring charge variants include cation exchange chromatography (CIEX), where the variants are quantified by dividing the area under the peak corresponding to the variant, e.g., acidic, basic, or neutral populations, by the cumulative area contained under all peaks in the sample spectrum. The change in charge variant population percentage between two samples, e.g., sample A and sample B, is calculated as the numerical difference in the respective population variant percentages, i.e., by subtracting the percentage of a particular variant, e.g., acidic, in sample B from the percentage of a particular variant, e.g., acidic, in sample A, or vice versa. In certain embodiments, the analysis can be similarly extended for all variants in the population.

[0059] Particles according to the present disclosure are circular. Circularity can serve as an indicator of the shape of a particle. Particles described herein can have a characteristic circularity, e.g., a relative shape that is substantially circular. This characteristic describes and defines the morphology of a particle based on its circularity. If a particle has a completely circular structure, the circularity is 1.0. Particles according to the present disclosure have a circularity 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, the circularity of substantially all of the particles is about 0.85 to about 1.00. In some embodiments, the circularity of substantially all of the particles is about 0.90 to about 1.00. In certain embodiments, the circularity of substantially all of the particles is about 0.95 to about 1.00. In certain embodiments, the circularity of substantially all of the particles is about 0.98 to about 1.00. In certain embodiments, the circularity of substantially all of the particles is about 1.00. The diameter and circularity of the particles can be determined by processing images observed under an electron microscope or the like or by a flow-type particle image analyzer. The circularity can also be determined by subjecting the particles to a circularity measurement and averaging the obtained values. For example, the circularity (circ) can be calculated using the following formula:

number

[0060] The term "perimeter" as used herein refers to the sum of all the borders of a closed planar figure or two-dimensional image. The term "area" as used herein refers to the cross-sectional area of ​​a two-dimensional image of a particle. The circularity of a particle can also be described as the ratio of the smallest dimension of the particle to its largest diameter. For a perfect circle, this ratio is 1. The circularity percentage can be calculated by multiplying the circularity by 100. The circularity can be calculated, for example, by measuring the aspect ratio using any software adapted to handle images, for example images obtained by microscopy, in particular scanning electron microscopy (SEM) or transmission electron microscopy (TEM). In some embodiments, a method for measuring particle circularity includes image analysis of a scanning electron micrograph of a particle, where the average roundness is calculated based on the cross-sectional shape of the particle projected onto the plane of the image. Such a roundness factor can be expanded to identify the corresponding circularity.

[0061] In certain embodiments of the present disclosure, the particles described herein have a surface morphology that is smooth, rather than bumpy or wrinkled. Those skilled in the art of the present disclosure can readily assess the surface morphology of the disclosed particles using routine and standard techniques.

[0062] In certain embodiments, the particles have a diameter of about 0.1 to about 1000 μm, e.g., about 0.1 to about 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, e.g., 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 micrographs of the particles, where the mean spherical radius or diameter can be calculated based on the cross-sectional area of ​​the particles projected onto the plane of the image.

[0063] As used herein, the term "dispersity index" (DI) is a parameter that characterizes the degree of heterogeneity of particle size distribution. Polydispersity index (PDI), "population dispersity" or "span", e.g., D10, D50, D90, can also refer to values ​​that indicate the width of particle size distribution. Particle size distribution is reported by D10, D50, D90, and mean particle size in μm, where the values ​​represent the percentage of particles smaller than the indicated D number, e.g., D10 particle size is the particle diameter where 10% of the mass is composed of particles with a diameter less than this value, D50 particle size is the particle diameter where 50% of the mass is composed of particles with a diameter less than this value, and D90 particle size is the particle diameter where 90% of the mass is composed of particles with a diameter less than this value. D10, D50, and D90 particle size distributions can be measured using a laser light scattering particle distribution meter.

[0064] Particle diameter and dispersibility can also affect injectability, e.g., syringeability. It is often recommended that particles be at least 3-10 times smaller than the inner diameter of the needle. Even if a small fraction of the particle population is larger than the inner diameter of the needle, they can clog the needle and waste the entire dosage. In certain embodiments of the present disclosure described herein, a high concentration of therapeutic biologic in the composition is achieved by mixing particles of various sizes.

[0065] As used herein, the terms "moisture" and "water" may be used interchangeably. In some embodiments, the moisture content of substantially all of the particles is less than about 7% by weight, such as less than about 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 of the particles have less than about 7% by weight moisture. In certain embodiments, substantially all of the particles have less than about 5% by weight moisture. In certain embodiments, substantially all of the particles have less than about 3% by weight moisture. In some embodiments, substantially all of the particles have less than about 1% by weight moisture. Exemplary methods for measuring moisture content include chemical titration methods, such as Karl Fischer titration with an oven. Various solvents, including water, may also be measured using weight loss methods with thermal excitation. Exemplary methods include thermogravimetric analysis coupled with infrared spectroscopy (TGA-IR) or gas chromatography flame ionization detector / mass spectrometry (GC-FID / MS).

[0066] In contrast to pores or porosity, the term "internal void space" or "internal voids" as used herein does not communicate with the surface of a solid (e.g., a particle) and does not contribute to the porosity or surface area. In some embodiments, substantially all of the particles have less than about 10% internal void space, e.g., less than about 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 the internal void space can be achieved by using focused ion beam scanning electron microscopy (FIB-SEM), which can be used to visualize "accessible" pores and "inaccessible" void space. Another method for determining the internal void space can be achieved by gas displacement pycnometer, a common analytical technique that uses gas displacement 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 an appropriate inert gas, and then expanded to another precise internal volume. The pressure before and after expansion is measured and used to calculate the sample volume. This volume is divided by the sample weight to obtain the gas displacement density. Cross sections of typical particles of the present disclosure show the absence of any internal void space as shown by FIB-SEM or by gas pycnometer using helium at a temperature of about 22° C., typically with a standard deviation of about 0.0005 g / cm 3 On average, it is about 1.3g / cm 3 For example, the internal void space can be calculated using the following formula: Internal void space = A v / A p , where A v is the total area of ​​void space, A pis the total area of ​​the particle. Cross-sections of exemplary particles of the present disclosure can be obtained and characterized in a number of 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 which are incorporated herein by reference in their entirety.

[0067] In some embodiments, substantially all of the particles have less than about 10% internal void space. In certain embodiments, substantially all of the particles have less than about 5% internal void space. In certain embodiments, substantially all of the particles have less than about 3% internal void space. In some embodiments, substantially all of the particles have less than about 1% internal void space. In certain embodiments, substantially all of the particles are substantially free of any internal void space.

[0068] In some embodiments, substantially all of the particles have greater than about 50% by weight of protein, e.g., therapeutic biologic, e.g., greater 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 biologic. In some embodiments, substantially all of the particles have greater than about 60% by weight of therapeutic biologic. In certain embodiments, substantially all of the particles have greater than about 70% by weight of therapeutic biologic. In some embodiments, substantially all of the particles have greater than about 80% by weight of therapeutic biologic. In certain embodiments, substantially all of the particles have greater than about 90% by weight of therapeutic biologic. In certain embodiments, substantially all of the particles have greater than about 95% by weight of therapeutic biologic. In certain embodiments, substantially all of the particles have greater than about 98% by weight of therapeutic biologic. In some embodiments, substantially all of the particles comprise greater than about 99% by weight of the therapeutic biologic.

[0069] The term "excipient" refers to an additive to a particle formulation that may be useful to achieve desired modifications to particle characteristics or morphology. 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 further comprise at least carbohydrates and amino acids. In some embodiments, substantially all of the particles further comprise at least surfactants and amino acids. In certain embodiments, substantially all of the particles further comprise at least one amino acid.

[0070] In some embodiments, the carbohydrate may be from the monosaccharide, disaccharide, oligosaccharide, or polysaccharide family. In some embodiments, the carbohydrate is dextran, trehalose, sucrose, agarose, mannitol, lactose, sorbitol, maltose, starch, alginate, xanthan, 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, sulfobutylether beta-cyclodextrin, methyl beta-cyclodextrin, or a combination thereof. In certain embodiments, the carbohydrate is trehalose, sucrose, sorbitol, or a combination thereof. 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.

[0071] The terms "pH adjusting agent" or "buffer species" or "buffering agent" are used interchangeably in the broadest sense herein. In some embodiments, substantially all of the particles include a pH adjusting agent. In some embodiments, substantially all of the particles include one or more pH adjusting agents. In certain embodiments, 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, glyclyglycine, monosodium glutamate, sodium hydroxide, potassium hydroxide, or a combination thereof. In certain embodiments, the pH adjusting agent is acetate, citrate, histidine, phosphate, succinate, hydrochloric acid, sodium hydroxide, or a combination thereof. In certain embodiments, the pH adjusting agent is histidine, hydrochloric acid, or a combination thereof. In certain embodiments, the pH adjusting agent 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 level is about 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. In certain embodiments, the pH level is about 5.0, 5.5, 6.0, 6.5, or 7.0.

[0072] 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 certain embodiments, the salt is sodium chloride.

[0073] In some embodiments, the chelating agent is edetate disodium, ethylenediaminetetraacetic acid, pentetic acid, or a combination thereof.

[0074] In some embodiments, the surfactants include, but are not limited to, (i) cationic surfactants such as cetyltrimethylammonium chloride, hexadecyltrimethylammonium chloride, benzalkonium chloride, benzethonium chloride, dioctadecyldimethylammonium bromide, (ii) anionic surfactants such as magnesium stearate, sodium stearate, sodium dodecyl sulfate, dioctyl sodium sulfosuccinate, sodium myreth sulfate, perfluorooctane sulfonate, alkyl ether phosphates, Aerosol-OT (sodium bis(2-ethylhexyl) sulfosuccinate), (iii) polysorbates, polyethylene glycol tert-octylphenyl ether, lecithin, alkylphenol ethoxylates, fatty alcohol ethoxylates (octaethylene glycol monododecyl ether, cocamide diethanolamine, poloxamers, glycerol monostearate, fatty amines, fatty acid esters of sorbitol (sorbitan monolaurate, Tween 80, Tween 20, nonionic surfactants such as poloxamers (nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), e.g., poloxamer 188, poloxamer 407, and (iv) zwitterionic surfactants such as cocamidopropyl hydroxysultaine and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS).In some embodiments, the surfactant is a polysorbate, magnesium stearate, sodium dodecyl sulfate, alkylphenol ethoxylate, glycerin, polyoxyethylated castor oil, docusate, sodium stearate, decyl glucoside, nonoxynol-9, cetyltrimethylammonium bromide, sodium bis(2-ethylhexyl)sulfosuccinate, lecithin, sorbitan esters, 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 combinations thereof. In certain embodiments, the surfactant is a polysorbate, docusate, 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, such as Tween 20, Tween 60, or Tween 80. In certain embodiments, the fatty acid ester of sorbitol is a sorbitan ester, such as 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 a sorbitan ester, polysorbate, poloxamer, polyethylene glycol tert-octylphenyl ether, lecithin, sodium stearate, magnesium stearate, or a combination thereof. In some embodiments, the surfactant is a polysorbate, a sorbitan ester, a poloxamer, or a combination thereof.

[0075] In some embodiments, the protein stabilizer is acetyl tryptophanate, caprylate, N-acetyl tryptophan, trehalose, polyethylene glycol (PEG), poloxamer, 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), poloxamer, 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, sulfobutylether beta-cyclodextrin, methyl beta-cyclodextrin, acetyltryptophanate, caprylate, N-acetyltryptophan, propylene glycol, glucose star polymer, silicone polymer, polydimethylsiloxane, carboxymethylcellulose, poly(glycolic acid), poly(lactic-co-glycolic acid), polylactic acid, polycaprolactone (PCL), polyvinylpyrrolidone (PVP), ficoll, dextran, or a combination thereof. In certain embodiments, the protein stabilizer is trehalose, cyclodextrin, hydroxypropyl beta-cyclodextrin, sulfobutylether 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 synonymously with the term "stabilizer" as used herein may be salts, carbohydrates, sugars, or amino acids, preferably carbohydrates or sugars that are recognized by authorities as suitable additives or excipients in pharmaceutical compositions.In certain embodiments, the 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 degradation of the protein, for example, during mixing and / or storage of the particulate material. Exemplary stabilizers include, but are not limited to, sugars, salts, hydrophobic salts, detergents, reducing agents, cyclodextrins, polyols, carboxylic acids, and amino acids. A "stable" formulation or composition as described herein refers to a particle formulation or composition comprising particles in which the protein retains an acceptable portion of its essential physical, chemical, or biological properties over an acceptable period of time. In the case of proteins, for example, 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 samples at several stages. These include, for example, before particle formation (evaluation of the aqueous feed solution), immediately after particle formation, and again after a storage period, where the storage is performed either in or in the absence of a suspension carrier medium.

[0076] Examples of emulsifiers suitable for use in the particles include, but are not limited to, lipophilic agents having an HLB of less than 7, such as 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-lactoesters of fatty acids; propylene glycol esters including propylene glycol monopalmitate, propylene glycol monostearate, and propylene glycol monooleate; sorbitan including sorbitan monostearate, sorbitan sesquioleate. esters; fatty acids including stearic acid, palmitic acid, and oleic acid, and soaps thereof; and mixtures thereof such as glyceryl monooleate, glyceryl dioleate, glyceryl monostearate, glyceryl distearate, glyceryl monopalmitate, and glyceryl dipalmitate; glyceryl-lactoesters of fatty acids; propylene glycol esters including propylene glycol monopalmitate, propylene glycol monostearate, and propylene glycol monooleate; sorbitan esters including sorbitan monostearate, 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 a 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 oleic glyceride, poloxamer, or combinations thereof. In some embodiments, the fatty acid ester of sorbitol is a 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.

[0077] In some embodiments, the preservative is phenol, m-cresol, benzyl alcohol, 2-phenyloxyethanol, chlorobutanol, neomycin, benzethonium chloride, gluteraldehyde, beta-propiolactone, or a combination thereof.

[0078] In certain embodiments, the amino acid is alanine, aspartic acid, cysteine, isoleucine, glutamic acid, leucine, methionine, phenylalanine, pyrrolysine, serine, selenocysteine, threonine, tryptophan, tyrosine, valine, asparagine, arginine, histidine, glycine, glutamine, proline, or various salts thereof (such as arginine hydrochloride, arginine glutamate, etc.), or combinations thereof. In some embodiments, the amino acid is alanine, aspartic acid, cysteine, isoleucine, glutamic acid, leucine, methionine, phenylalanine, pyrrolysine, serine, selenocysteine, threonine, tryptophan, tyrosine, valine, asparagine, arginine, histidine, glycine, glutamine, proline, or combinations thereof. Yet, in some embodiments, the amino acid is 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 acid is arginine and histidine. In certain embodiments, the amino acid is histidine.

[0079] 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 parahydroxybenzoic acid. In certain embodiments, the bactericide is benzalkonium chloride (a cationic surfactant), hypochlorite, peroxide, alcohol, a phenolic compound (e.g., carbolic acid), benzyl benzoate, or a combination thereof. In certain embodiments, the bactericide is benzalkonium chloride or benzyl benzoate. In certain embodiments, the fungicide is acibenzolar, 2-phenylphenol, anilazine, carvone, natamycin, potassium azide, or a combination thereof.

[0080] 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.

[0081] A plurality of particles comprising at least one protein, e.g., a therapeutic biologic described herein, can be used in a number of ways, as well as in a number of different applications, such as those described in, for example, International Application Nos. PCT / US2017 / 063150 (Publication No. WO2018 / 098376), PCT / US2018 / 043774 (Publication No. WO2019 / 023392), PCT / US2019 / 033875 (Publication No. WO2019 / 226969), PCT / US2020 / 15957 (Publication No. WO2020 / 160323), PCT / US2020 / 0 The particles can be prepared and characterized in any of the methods for forming particles disclosed in PCT / US2021 / 16878 (WO2021 / 158959), PCT / US2021 / 018806 (WO2021 / 168271), PCT / US2021 / 027755 (WO2021 / 212019), and PCT / US2022 / 072755 (WO2022 / 256840), the contents of each of which are incorporated herein by reference in their entirety.

[0082] Although each of the elements of the present disclosure are described herein as including multiple embodiments, unless otherwise indicated, it is to be understood that each of the embodiments of a given element of the present disclosure can be used with each of the embodiments of the other elements of the present disclosure, and each such use is intended to form a separate embodiment of the present disclosure.

[0083] 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 the information known to those of skill in the art, and will be understood by those of skill in the relevant art to be able to make such modifications without departing from the scope of the disclosure or any embodiment thereof.

[0084] Pharmaceutical Compositions of the Present Disclosure Injectable particle suspensions can exhibit variations in settling, which can affect the use or administration of drug compositions. In particular, injectable particle suspensions with high therapeutic biological agent concentrations, low volumes, and low syringe forces can settle or settle out of the suspension medium over a period of time, thus requiring premixing or resuspension before injection. Settling of high particle concentrations at low delivery volumes can also lead to high injection forces and propagate degradation of the therapeutic biological agent in the composition. As used herein, the term "settling" or "sedimentation" refers to the process in which particles settle or densely settle to the bottom of a container closure, such as a vial, cartridge, syringe, portable drug delivery injection device, etc. Settling of densely settled particles generally leads to excessively high injection forces or syringe blockages, requiring manual agitation or premixing before administration of the composition.

[0085] Flocculation or "particle agglomeration" is a phenomenon that results from the interrelationship between interfacial chemistry and environmental conditions that govern particle-particle interactions. Flocculation volume, as used herein, is a measure of the amount of particle agglomerates occupied by a particle dispersion, expressed as a percentage of the total fluid volume. More specifically, the flocculation volume (F) is the ratio of the volume of particle agglomerates (Vu) to the total fluid volume (Vo). The flocculation volume can decrease over time based on the settling characteristics of the particle agglomerates. In a flocculated suspension, particles undergo collisions that lead to particle agglomerates or agglomerates, which increase in size, e.g., the flocculation volume increases. The term "agglomerates" refers to clusters of particles that are loosely cohesive. The loose network of particle agglomerates maintains sufficient interparticle distance to prevent particle settling, which can lead to high injection forces. When the flocculation volume is equal to one, the yield stress (the stress at which the flocculation volume undergoes deformation) can be measured to indicate the shear stress that is required to create flow in the suspension. Measurements for yield stress can be obtained by a parallel plate rheometer (ANTON PAAR™ MCR 92). An increase in yield stress indicates an increase in agglomeration. Other approaches for measuring agglomeration include, but are not limited to, laser diffraction, relaxation NMR, microscopy, and small angle scattering.

[0086] In the compositions disclosed herein, the agglomeration process can be controlled by the use of flocculants. Flocculants, or flocculants, are chemicals that promote aggregation by agglomerating suspended particles in a liquid, thereby forming agglomerates. In non-aqueous particle suspensions, the breakup and redispersion of agglomerates is governed by the shear stress imparted to the agglomerated particle system. Shear stress is caused by forces acting on the surface of the material, causing deformation. In these systems, shear can result from the movement of particles or agglomerates through velocity gradients in the non-aqueous suspension medium or near any interface (e.g., container closures, other particles, etc.). Shear stress can be, for example, mechanically introduced into the agglomeration volume and can be experimentally controlled.

[0087] The compositions disclosed herein include a flocculating agent and are generally high concentration, low volume, and low syringe force injectable particle suspensions of therapeutic biological agents that allow for administration without the need for manual agitation or premixing prior to injection or administration of the composition. The compositions advantageously maintain a stable flocculation volume that avoids particle settling that can result in clogging of syringes, ambulatory drug delivery injection devices, or orally administered liquid syringe capsules.

[0088] In certain embodiments, the present disclosure relates to a composition, e.g., a pharmaceutical composition, comprising a plurality of particles suspended in a pharma- ceutically acceptable liquid carrier, wherein the particles (e.g., substantially all of the particles) comprise a therapeutic biologic (e.g., a therapeutic biologic disclosed herein), and a flocculating agent (e.g., a flocculating agent disclosed herein).

[0089] The phrase "pharmaceutically acceptable liquid carrier" as used herein means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid, diluent, excipient, or solvent. Each carrier must be "acceptable" in the sense of being compatible with the other components of the composition and not harmful to the patient. Those skilled in the art can use inert substances in the particles to dilute or increase the volume of the therapeutic biological product. These diluents can include carbohydrates, especially trehalose, mannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans, and starch. Certain inorganic salts, including calcium triphosphate, magnesium carbonate, and sodium chloride, can also be used as bulking agents. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress, and Avicell.

[0090] In some embodiments, the pharmaceutical composition for administration includes a non-aqueous solution of the active therapeutic biological agent in water-soluble form. Preferably, the suspension of the active therapeutic biological agent 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. The appropriate viscosity can be maintained by maintaining the required particle size in the case of injection and by the use of flocculating agents. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

[0091] The present disclosure includes the use of "pharmaceutically acceptable salts" or "salts" of therapeutic biologics in the compositions of the present disclosure. The term "pharmaceutically acceptable salts" or "salts" as used herein includes salts derived from inorganic or organic acids, including, for example, 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, pharmaceutically acceptable salt forms can include forms in which the ratio of salt-containing molecules is not 1:1. For example, a salt can include more than one inorganic or organic acid molecule per molecule of base, such as two hydrochloric acid molecules per molecule of therapeutic biologic. As another example, a salt may contain less than one inorganic or organic acid molecule per molecule of base, such as two molecules of a therapeutic biologic per molecule of tartaric acid. In certain embodiments, contemplated salts of the present disclosure include, but are not limited to, arginine, benentamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, lH-imidazole, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, or zinc salts. In some embodiments, contemplated salts of the present disclosure include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts. In further embodiments, contemplated salts of the present disclosure include, but are not limited to, alkyl, dialkyl, trialkyl, or tetraalkyl ammonium salts. Pharmaceutically acceptable salts can also exist as various solvates, for example with water or ethanol. Mixtures of such solvates can also be prepared. In some embodiments, the proteinaceous compositions can be formulated in a neutral or salt form.Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) which are formed with inorganic acids, such as, for example, hydrochloric or phosphoric acids, or organic acids, such as acetic, oxalic, tartaric, mandelic, etc. Salts formed with the free carboxyl groups can also be derived from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine, and the like.

[0092] In some embodiments, the particles can be suspended in a non-aqueous liquid carrier, thereby forming a non-aqueous pharma- ceutically acceptable composition.Importantly, the process of producing a non-aqueous composition having at least one therapeutic biological agent does not significantly alter the structure or biological activity of the biological agent 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.

[0093] In some embodiments, the organic solvent is benzyl benzoate, coconut oil, cottonseed oil, fish oil, grapeseed oil, hazelnut oil, hydrogenated vegetable oil, olive oil, palm seed oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, sunflower oil, walnut oil, corn oil, acetone, ethyl acetate, ethyl lactate, dimethylacetamide, dimethylisosorbide, dimethylsulfoxide, glycofurol, diglyme, methyl tert-butyl ether, N-methylpyrrolidone, perfluorodecalin, polyethylene glycol, 2-pyrrolidone, tetrahydrofurfuryl alcohol, diglycerides, trigylcerides, medium chain triglycerides (MCT), caproic acid, caprylic acid, capric acid, lauric acid, ethyl laurate, triglycerides of fractionated vegetable fatty acids C8 and C10, propylene glycol of saturated vegetable fatty acids C8 and C10. Choline diesters (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 such as 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, ethyl macadamiatemacadamiate), 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, ethylhexyl acetate, propylene glycol dicaprylate, caprylic triglyceride, ethyl linoleate, ethyl linolenate, or combinations thereof. Medium chain triglycerides (MCTs) contain fatty acids with chain lengths of 6 to 12 carbon atoms. They include caproic acid (C6), caprylic acid (C8), capric acid (C10), and lauric acid (C12). In some embodiments, the organic solvent is ethyl oleate, diglyceride, triglyceride, miglyol, ethyl macadamiate, ethyl caprate, diethyl succinate, diethylene glycol monoethyl ether, propylene glycol dicaprylate, caprylic triglyceride, ethyl linoleate, ethyl linolenate, medium chain triglyceride (MCT), sesame oil, propylene glycol diesters of saturated vegetable fatty acids C8 and C10 (PGD), triacetin, or combinations thereof. In certain embodiments, the organic solvent is ethyl oleate, ethyl caprylate, propylene glycol dicaprylate, diglyceride, triglyceride, sesame oil, propylene glycol diesters of saturated vegetable fatty acids C8 and C10 (PGD), triacetin, or combinations thereof. In some embodiments, the organic solvent is ethyl oleate and sesame oil.

[0094] In some embodiments, the concentration of the therapeutic biologic in the composition disclosed herein is greater than about 250 mg / mL, e.g., 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 biologic in the composition is greater than about 800 mg / mL. In some embodiments, the concentration of the therapeutic biologic in the composition is greater than about 400 mg / mL. In certain embodiments, the concentration of the therapeutic biologic in the composition is greater than about 500 mg / mL. In some embodiments, the concentration of the therapeutic biologic in the composition is greater than about 600 mg / mL. In certain embodiments, the concentration of the therapeutic biologic in the composition is greater than about 700 mg / mL.

[0095] In the compositions described herein, the agglomeration process can be controlled by the use of a flocculating agent, which can allow administration of the particle composition without the need for manual stirring or premixing prior to injection or administration. The flocculating agent can be non-ionic or ionic. In general, ionic flocculating agents interact with charged particles, e.g., anionic flocculating agents interact with positively charged particles and cationic flocculating agents interact with negatively charged particles. In some embodiments, the flocculating agent is ionic. In some embodiments, the ionic flocculating agent is magnesium stearate, sodium dodecyl sulfate, sodium stearate, cetyltrimethylammonium bromide, lecithin, or a combination thereof. In certain embodiments, the flocculating agent is non-ionic. As disclosed herein, non-ionic flocculating agents interact with charged particles. In certain embodiments, the non-ionic flocculating agent is a polysorbate (polysorbate 80, polysorbate 60, polysorbate 20, e.g., Tween 80, Tween 60, Tween 20), an alkylphenol ethoxylate, glycerol, polyoxyethylated castor oil, docusate, 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 oleic glyceride, poloxamer, or a combination thereof. In some embodiments, the non-ionic flocculating agent is a polysorbate, an alkylphenol ethoxylate, a sorbitan ester, a poloxamer, or a combination thereof. In certain embodiments, the non-ionic flocculating agent is a polysorbate or a sorbitan ester. In some embodiments, the addition of the flocculant to the particle suspension does not substantially increase the viscosity of the composition. As disclosed herein, the addition of the flocculant to the particle suspension increases the flocculation volume of the particle suspension (see, for example, FIG. 4 and FIG. 6). In certain embodiments, the addition of the flocculant to the particle suspension increases the stability of the flocculation volume for at least one month (see, for example, Example 6).In some embodiments, the addition of a flocculating agent to a suspension of particles reduces syringe force (see, e.g., Examples 16 and 17). In some embodiments, the addition of a flocculating agent to a suspension of particles improves the pharmacokinetics (PK) of administration (e.g., subcutaneous administration) of a suspension of microparticles (e.g., Ab microparticles), which exhibits higher bioavailability than administration (e.g., subcutaneous administration) of an aqueous Ab solution (see, e.g., FIG. 11). In some embodiments, the addition of a flocculating agent to a suspension of particles increases the stability of the protein in the particles (see, e.g., FIG. 3).

[0096] In some embodiments, the compositions described herein use a concentration of the flocculant 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 concentration of the flocculant in the composition is less than about 10 mg / mL. In some embodiments, the concentration of the flocculant in the composition is less than about 5 mg / mL. In some embodiments, the concentration of the flocculant in the composition is less than about 3 mg / mL. In certain embodiments, the concentration of the flocculant in the composition is less than about 1 mg / mL. In some embodiments, the concentration of the flocculant in the composition is less than about 0.1 mg / mL. In certain embodiments, the concentration of the flocculant in the composition is less than about 0.01 mg / mL.

[0097] Viscosity can play an important role in the handling and administration of injectable products. Highly viscous drug products can be difficult to deliver through needles (e.g., 27-gauge needles) because suspension products require more force to actuate injection devices, such as syringes, portable drug delivery injection devices, or orally administered liquid injector capsules. Alternatively, using larger gauge needles or requiring longer injection times can reduce patient compliance with therapy.

[0098] The term "viscosity" is used to describe the property of a fluid that acts 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) at a specified shear rate at 25° C., for example, an AR-G2 rheometer (TA Instruments, USA). In certain embodiments, viscosity is measured at a shear rate in the Newtonian region. In some embodiments, viscosity is measured at a shear rate of 100 s -1 or higher shear rates, e.g., 1000 s -1 , or 1000s -1 More than or equal to 10,000 -1 As used herein, the term "low viscosity" describes a composition, e.g., a liquid carrier, having a viscosity of less than about 100 mPa·s.

[0099] In some embodiments, the composition has a molecular weight of less than about 200 mPa·s, less than about 150 mPa·s, less than about 125 mPa·s, less than about 100 mPa·s, less than about 95 mPa·s, less than about 90 mPa·s, less than about 85 mPa·s, less than about 80 mPa·s, less than about 75 mPa·s, less than about 70 mPa·s, less than about 65 mPa·s, less than about 60 mPa·s, less than about 55 mPa·s, less than about 50 mPa·s, less than about 45 mPa·s, less than about 40 mPa·s, less than about 35 mPa·s, less than about 30 mPa·s, less than about 25 mPa·s, less than about 20 mPa·s , less than about 19 mPa·s, less than about 18 mPa·s, less than about 17 mPa·s, less than about 16 mPa·s, less than about 15 mPa·s, less than about 14 mPa·s, less than about 13 mPa·s, less than about 12 mPa·s, less than about 11 mPa·s, less than about 10 mPa·s, less than about 9.5 mPa·s, less than about 9 mPa·s, less than about 8.5 mPa·s, less than about 8 mPa·s, less than about 7.5 mPa·s, less than about 7 mPa·s, less than about 6.5 mPa·s, less than about 6 mPa·s, less than about 5.5 mPa·s, or less than about 5 mPa·s (1 millipascal second). Viscosity can also be controlled using a mixture of liquids. The units "mPa·s" and "cP" are used interchangeably in the broadest sense herein.

[0100] 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 certain embodiments, 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 certain embodiments, the composition has a viscosity of less than about 30 mPa·s. In certain embodiments, 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 certain embodiments, 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 certain embodiments, the composition has a viscosity of less than about 5 mPa·s.

[0101] As described herein, a higher aggregate volume indicates increased agglomeration or less particle settling, and thus improved injection force performance due to more uniform dispersion of particles in the liquid carrier. In some embodiments, the composition has an aggregate 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 an aggregate volume of more than about 70% after initial mixing. In certain embodiments, the composition has an aggregate volume of more than about 80% after initial mixing. In certain embodiments, the composition has an aggregate volume of more than about 85% after initial mixing. In certain embodiments, the composition has an aggregate 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 to create the aggregate volume.

[0102] In some embodiments, the aggregate 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 container-closed storage conditions below about 40° C. In some embodiments, the aggregate volume is reduced by less than about 7% after at least one month under container-closed storage conditions below about 40° C. In certain embodiments, the aggregate volume is reduced by less than about 5% after at least one month under container-closed storage conditions below about 40° C. In some embodiments, the aggregate volume is reduced by less than about 3% after at least one month under container-closed storage conditions below about 40° C. In certain embodiments, the aggregate volume is reduced by less than about 1% after at least one month under container-closed storage conditions below about 40° C. In some embodiments, the plurality of particles and the aggregate agent 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.

[0103] In certain embodiments, insoluble particulate matter having a characteristic size of about 100 μm or greater that persists upon dissolution in an aqueous liquid is referred to as a visible particle (VP). In certain embodiments of the present disclosure described herein, the composition is 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 that distributes to the extracellular compartment, such as extracellular fluid, interstitial fluid, intravascular fluid (blood, plasma, and lymph), and cerebrospinal fluid.

[0104] In some embodiments, insoluble particulate matter having a characteristic size of about 1 μm to about 100 μm that persists upon dissolution in an aqueous liquid is referred to as sub-visible particles (SvP). SvP is present in an amount of about 0 to about 100,000,000 per mL, for example, about 0 to about 10,000,000 per mL, about 0 to about 1,000,000 per mL, about 0 to about 500,000 per mL, about 0 to about 100,000 per mL, about 0 to about 50,000 per mL, about 0 to about 10,000 per mL, about 0 to about 6,000 per mL, about 0 to about 1,000 per mL, about 0 to about 600 per mL, about 0 to about 250 per mL, about 0 to about 100 per mL, about 0 to about 60 per mL, or about 0 to about 10 per mL. In some embodiments, the count of particles having a characteristic size of 10 μm or greater is about 0 to about 6,000 per mL, e.g., about 0 to about 1,000 per mL, about 0 to about 100 per mL, about 0 to about 10 per mL, about 0 to about 5 per mL, about 0 to about 3 per mL, or about 0 to about 1 per mL. In certain embodiments, the count of particles having a characteristic size of 25 μm or greater is about 0 to about 600 per mL, e.g., about 0 to about 100 per mL, about 0 to about 10 per mL, about 0 to about 3 per mL, about 0 to about 1 per mL, about 0 to about 0.5 per mL, or about 0 to about 0.1 per mL. Exemplary methods for measuring SvP include analysis of the therapeutic biologic with a Coulter Counter, HIAC Royco, or microfluidic imaging system after reconstitution and dilution of the therapeutic biologic to a standard concentration, e.g., about 100 mg / mL or about 1 mg / mL. In some embodiments, the composition, when dissolved in an aqueous liquid, has a concentration of about 0 to about 100,000,000 insoluble sub-visible particles (SvP) per mL of particles greater than about 10 μm. In certain embodiments, the composition, when dissolved in an aqueous liquid, has a concentration of about 0 to about 6000 insoluble sub-visible particles (SvP) per mL of particles greater than about 10 μm. In certain embodiments, the composition, when dissolved in an aqueous liquid, has a concentration of about 0 to about 600 insoluble sub-visible particles (SvP) per mL of particles greater than about 25 μm.In certain embodiments, the composition, when dissolved in an aqueous liquid, is substantially free of insoluble sub-visible particles (SvP).

[0105] In certain embodiments, the composition, when dissolved in an aqueous liquid, has a concentration of insoluble sub-visible particles (SvP) having a characteristic size greater than about 10 μm per mL from about 0 to about 100,000,000 per mL. In some embodiments, the composition, when dissolved in an aqueous liquid, has a concentration of insoluble sub-visible particles (SvP) having a characteristic size greater than about 10 μm per mL from about 0 to about 6000 per mL. In some embodiments, the composition, when dissolved in an aqueous liquid, has a concentration of insoluble sub-visible particles (SvP) having a characteristic size greater than about 25 μm per mL 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.

[0106] In some embodiments, insoluble particulate matter having a characteristic size of about 100 nm to about 1 μm that persists upon dissolution in an aqueous liquid is referred to as a submicron particle (SMP) and is often known as a nanoparticle. The presence of such SMP is believed to contribute to immunogenicity and should therefore be avoided to minimize such effects. Quantitatively, SMPs range from about 0 to 5×10 per mL. 12 For example, about 0 to about 0.5 × 10 per mL 12 , about 0 to about 50 × 10 per mL 9 , about 0 to about 10 × 10 per mL 9 , about 0 to about 5 × 10 per mL 9 , about 0 to about 0.5 × 10 per mL 9 , about 0 to about 50 × 10 per mL 6 , about 0 to about 1 × 10 per mL 6, about 0 to about 500,000 per mL, about 0 to about 200,000 per mL, about 0 to about 100,000 per mL, about 0 to about 10,000 per mL, about 0 to about 5000 per mL, or about 0 to about 1000 per mL. Exemplary methods for quantitatively measuring SMP include analysis of the therapeutic biologic by NanoSight, a microfluidic imaging system, asymmetric field flow fractionation coupled to multi-angle laser light scattering (AF4 MALS), dynamic light scattering (DLS), or FLOWCAM™ imaging after reconstitution and dilution of the therapeutic biologic to a standard concentration, e.g., about 100 mg / mL, about 1 mg / mL, or about 1 μg / mL. Qualitatively, the SMP is in a range that corresponds to the starting monomeric therapeutic biologic solution. In certain embodiments, the composition is substantially free of submicron particles (SMPs) when dissolved in an aqueous liquid.

[0107] The disclosure described herein relates to a highly concentrated composition comprising a flocculating agent and a plurality of particles suspended in a pharma- ceutically acceptable liquid carrier, the particles (e.g., substantially all of the particles) comprising at least one therapeutic biological agent, the composition having substantially similar immunogenicity when dissolved in water, a buffer, or other physiologically relevant aqueous liquid, e.g., a biological fluid within a patient's body, compared to a similar aqueous composition comprising a monomeric therapeutic biological agent. As used herein, "physiologically relevant" conditions as may be encountered within a mammal or human may be applied. One skilled in the art will be able to determine the most appropriate set of conditions for testing according to the end use of the composition described herein. In some embodiments, the composition has substantially similar immunogenicity when dissolved in an aqueous liquid, compared to an aqueous composition comprising at least one therapeutic biological agent in a soluble form, e.g., in a monomeric 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.

[0108] As disclosed herein, the term "immunogenicity" refers to the induction of an immune response by an injected composition of a therapeutic biologic (antigen), while "antigenicity" refers to the reaction of a composition of a therapeutic biologic with pre-existing antibodies. Taken together, antigenicity and immunogenicity are referred to as "immunoreactivity". In certain embodiments, the composition has substantially similar immunogenicity compared to an aqueous composition comprising at least one therapeutic biologic in a soluble form, e.g., in a monomeric form. In certain embodiments, the composition is substantially non-immunogenic when subjected to the presence of a non-immune response, e.g., based on a repeated dose anti-drug antibody immunogenicity analysis (see, e.g., Example 8).

[0109] The ratio between the toxicity and therapeutic effect of a particular composition, e.g., human IgG, is its therapeutic index, LD 50 (lethal dose of compound in 50% of the population) and ED 50 (the amount of compound effective in 50% of the population). Compositions that exhibit a high therapeutic index are preferred. Therapeutic index data obtained from cell culture assays and / or animal studies, e.g., human IgG, can be used in formulating various dosages for use in humans and are known in the art. The dosage of such compounds is preferably at least about ED with little or no toxicity. 50 The plasma concentration range includes: 0.1 to 0.5% by weight of the compound; 0.1 to 0.5% by weight of the compound; 0.2 to 0.5% by weight of the compound; 0.3 to 0.5% by weight of the compound; 0.4 to 0.6% by weight of the compound; 0.5 to 0.7% by weight of the compound; 0.6 to 0.8% by weight of the compound; 0.8 to 0.9% by weight of the compound; 0.9 ...

[0110] In some embodiments, the composition has substantially similar toxicity compared to an aqueous composition comprising at least one therapeutic biologic in soluble form, e.g., monomeric form. In some embodiments, the composition has reduced toxicity compared to an aqueous composition comprising at least one therapeutic biologic in soluble form, e.g., monomeric form. In certain embodiments, the composition is substantially non-toxic, e.g., based on local tolerability and clinical observation analysis (see, e.g., Example 7).

[0111] In certain embodiments according to the present disclosure described herein, the particle composition has improved stability of the therapeutic biological agent compared to an aqueous composition comprising at least one therapeutic biological agent in a soluble form, e.g., monomeric form (see, e.g., FIG. 3). A "stable" composition is one in which all therapeutic biological agents therein essentially retain their physical and / or chemical stability and / or biological activity upon storage, e.g., in a container closure, at the intended storage temperature, e.g., 4-40°C. It is desirable for the composition to essentially retain its physical and chemical stability, as well as its biological activity, upon storage. The storage period is generally selected based on the intended shelf life of the composition. Additionally, the composition should be stable after freezing (e.g., to -70°C) and thawing the composition, e.g., after 1, 2 or 3 cycles of freezing and thawing. A variety of analytical techniques for measuring protein stability are available in the art and are 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 for a selected period of time. Stability can be qualitatively and / or quantitatively assessed in a variety of different ways, including assessing aggregate formation (e.g., using size-exclusion chromatography and / or by visual inspection); by assessing charge heterogeneity using cation exchange chromatography or capillary zone electrophoresis; amino- or carboxy-terminal sequence analysis; mass spectrometry; SDS-PAGE analysis comparing reduced and intact antibodies; peptide map (e.g., trypsin or LYS-C) analysis; assessing antibody biological activity or antigen-binding function, and the like. In some embodiments, the therapeutic biologic in the composition is stable for at least one month. In some embodiments, the therapeutic biologic in the composition is stable for at least two months.In certain embodiments, the therapeutic biologic in the composition is stable for at least 3 months. In certain embodiments, the therapeutic biologic in the composition is stable at 40° C. for at least 3 months.

[0112] In some embodiments, the methods disclosed herein further comprise administering a pharma- tically effective amount of at least one hyaluronan degrading agent, which can be administered simultaneously, sequentially, or intermittently with the composition. Subcutaneous tissue or extracellular matrix consists of a network of fibrous proteins embedded in a viscoelastic gel of glycosaminoglycans. Hyaluronan is the predominant glycosaminoglycan of subcutaneous tissue. Hyaluronan is secreted by fibroblasts into the interstitium as a viscous polymer, which is then degraded locally in the lymph by the action of hyaluronidase. Glycosaminoglycans are complex linear polysaccharides of the extracellular matrix, characterized by a repeating disaccharide structure of N-substituted hexosamines and uronic acids, as in the case of hyaluronan.

[0113] Hyaluronan degrading agents can enhance subcutaneous administration of a composition comprising a plurality of particles and aggregating agents, for example, by enhancing and / or increasing the volume of the composition administered by injection, thereby improving the absorption of the therapeutic biological agent. The use of hyaluronan degrading agents, for example, hyaluronidase, can improve the subcutaneous administration of a therapeutic biological agent into the systemic circulation through the reversible hydrolysis of hyaluronan, for example, the reversible degradation of hyaluronan. The 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 into the subcutaneous tissue (see, for example, Example 9). In addition, the degradation of hyaluronan temporarily reduces the viscosity of the subcutaneous tissue, promoting the dispersion of injected liquids and facilitating their absorption. The effect of hyaluronidase is local and reversible, with complete reconstitution of the hyaluronan tissue occurring within 24-48 hours. See, e.g., Frost, GI, "Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration", Expert Opinion on Drug Delivery, 2007;4:427-440. Increased permeability of subcutaneous tissue due to degradation of hyaluronan correlates with the effectiveness of hyaluronidases for their ability to increase the dispersion and absorption of compositions containing multiple particles administered simultaneously, sequentially, or intermittently. In certain embodiments, the methods described herein further comprise administering at least one hyaluronan degrading agent.

[0114] In some embodiments, the methods of the disclosure further comprise administering at least one hyaluronan degrading agent, for example, a pharma- tically effective amount of at least one hyaluronan degrading agent administered simultaneously, sequentially, or intermittently with the composition. In certain embodiments, the hyaluronan degrading agent is a hyaluronidase enzyme, also referred to herein as hyaluronidase. In some embodiments, the hyaluronidase is a soluble neutral active hyaluronidase. In certain embodiments, the hyaluronidase is a mammalian hyaluronidase. In certain embodiments, the mammalian hyaluronidase is a human hyaluronidase. In certain embodiments, the human hyaluronidase is a recombinant human hyaluronidase, for example, rHuPH20. Certain recombinant human hyaluronidases suitable for use in the compositions disclosed herein are commercially available, for example, rHuPH20 from Halozyme Therapeutics (San Diego, Calif.).

[0115] According to certain embodiments of the present disclosure, the hyaluronan degrading agent can be administered simultaneously with the composition, sequentially, or intermittently. As used herein, the term "simultaneous" or "near simultaneous" administration refers to simultaneous administration of both components administered at the same time, e.g., the composition and the hyaluronan degrading agent. In some embodiments, the hyaluronan degrading agent and the composition are administered as a single composition, e.g., co-formulated. In some embodiments, the hyaluronan degrading agent and the composition are administered as separate formulations. However, one component may be administered within minutes or hours, e.g., in the same medical appointment or doctor's visit. Such administration is referred to as "sequential" administration. In certain embodiments, sequential administration refers to sequential administration of the hyaluronan degrading agent and the composition described herein. In certain embodiments, the hyaluronan degrading agent is administered first, followed by administration of one or more doses of the composition. In certain embodiments, the components may be administered intermittently, as co-formulations or separate formulations. In certain embodiments, the hyaluronan degrading agent is administered first.

[0116] Methods of the Disclosure When clinical applications of therapeutic biologics, such as therapeutic compositions containing mAbs, are made, it will generally be beneficial to prepare pharmaceutical or therapeutic compositions that are appropriate for the intended use. In certain embodiments, a pharmaceutical composition may, for example, contain at least about 0.1% of an active therapeutic biologic. In some embodiments, the active therapeutic biologic may comprise from about 2% to about 99%, or from about 50% to about 99%, by weight of the unit, and any range derivable therein.

[0117] As described herein, CD20 (also known as Bp35) is a B-lymphocyte-restricted differentiation antigen that is expressed during early pre-B cell development and remains until plasma cell differentiation. Because this antigen is expressed at very high density on the surface of malignant B cells, i.e., B cells whose unabated 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's lymphoma (NHL). Rituximab acts by binding to the CD20 antigen on B cells, thereby resulting in lysis of the B cells by a mechanism believed to involve complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). In certain embodiments, the therapeutic biologic is an antibody. In certain embodiments, the antibody is an anti-CD20 antibody.

[0118] Provided herein are compositions and methods for treating a disease or condition in a subject in need thereof, comprising administering to the subject a pharma- tically effective amount of a composition comprising a plurality of particles suspended in a pharma- tically acceptable liquid carrier, the particles (e.g., substantially all of the particles) comprising at least one therapeutic biologic or salt thereof, and an aggregating agent, wherein the concentration of the aggregating agent in the composition is less than about 50 mg / mL. In some embodiments, the concentration of the therapeutic biologic or salt thereof in the composition is greater than about 250 mg / mL. In certain embodiments, provided herein is a method for treating cancer, an inflammatory disease, or an immune disease in a subject in need thereof, comprising administering to the subject a pharma- tically effective amount of a composition comprising a plurality of particles suspended in a pharma- tically acceptable liquid carrier, the particles (e.g., substantially all of the particles) comprising at least one therapeutic biologic or salt thereof, and an agglutinating agent, the concentration of the agglutinating agent in the composition being less than about 50 mg / mL, and the concentration of the therapeutic biologic or salt thereof in the composition being greater than about 250 mg / mL. Also provided herein is a method for administering a pharma- tically effective composition comprising a plurality of particles suspended in a pharma- tically acceptable liquid carrier, the particles (e.g., substantially all of the particles) comprising at least one therapeutic biologic or salt thereof, and an agglutinating agent, the concentration of the agglutinating agent in the composition being less than about 50 mg / mL, and the concentration of the therapeutic biologic or salt thereof in the composition being greater than about 250 mg / mL.

[0119] The term "treat" or "treating" or "treatment" generally refers to a therapeutic treatment regimen, e.g., to treat, reverse, and / or downregulate a disease or condition. As used herein, "treat" or "treating" means administering, internally or externally, a therapeutic biologic, such as a composition containing any of the antibodies or antigen-binding fragments thereof of the present disclosure, to a subject having, or suspected of having, one or more disease symptoms for which the biologic has therapeutic or prophylactic activity. Typically, the therapeutic biologic is administered in an amount effective to alleviate one or more disease symptoms in the treated subject or population, whether by inducing regression of such symptom(s) or inhibiting its progression to any clinically measurable extent. The amount of therapeutic biologic that is effective to alleviate any particular disease symptom may vary depending on factors such as the condition, age, and weight of the subject or patient, as well as the ability of the therapeutic biologic to elicit a desired response in the subject or patient. Whether a disease symptom is alleviated may be assessed by any clinical measurement typically used by a physician or other skilled health care provider to assess the severity or progression of the symptom. The term further includes the postponement of the onset of symptoms associated with the disorder and / or the reduction in the severity of the symptoms of such a disorder. The term further includes the amelioration of existing uncontrolled or undesirable symptoms, the prevention of additional symptoms, and the amelioration or prevention of the underlying causes of such symptoms. Thus, the term indicates that a beneficial result has been conferred on a human or animal subject having a disorder, disease, or condition, or having the potential to develop such a disorder, disease, or condition. As used herein, "treatment" refers to therapeutic treatment and diagnostic applications as it applies to a human or veterinary subject. "Treatment" encompasses contacting a human or animal subject with a therapeutic biologic of the present disclosure, e.g., an antibody or antigen-binding fragment, as it applies to a human or veterinary subject. Subjects in need of treatment for cancer include those already having a benign, precancerous, or non-metastatic tumor, and those to prevent the appearance or recurrence of cancer.In some embodiments, the purpose or outcome of treating or treatment may be to reduce the number of cancer cells, reduce the size of the primary tumor, inhibit (i.e., slow to some extent, and preferably stop) cancer cell invasion into peripheral organs, inhibit (i.e., slow to some extent, and 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 efficacy of treatment may be measured by assessing the duration of survival, time to disease progression, response rate (RR), duration of response, and / or quality of life. The terms "prevent" or "preventing" or "prevention" as used herein refer to any action of inhibiting or delaying the onset of a disease or condition in a subject in need thereof, including administering to the subject a pharma- ceutical effective amount of a composition according to the present disclosure. In certain embodiments of the present disclosure, "treating" a subject suffering from a disease or condition is intended to include, but is not limited to, (i) slowing, halting, or reversing the progression of the disease or condition; (ii) slowing, halting, or reversing the progression of 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 symptoms of the disease or condition.

[0120] 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 1, hereditary nonpolyposis 2, hereditary nonpolyposis 3, hereditary nonpolyposis 6; colorectal cancer, hereditary nonpolyposis 7, small intestine and / or large intestine cancer, esophageal cancer, callus 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 type 1), liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma, hepatocellular carcinoma), bladder cancer, embryonal rhabdomyosarcoma, germ cell tumors, trophoblastic tumors, testicular germ cell tumors, ovary, uterus, epithelial ovarian immature teratoma, sacrococcygeal tumors, choriocarcinoma, placental trophoblastic tumors, epithelial adult tumors, ovarian cancer, serous ovarian cancer, ovarian sex cord tumors, cervical cancer, small cell and non-small cell lung cancer, nasopharyngeal, breast cancer (e.g., ductal carcinoma, invasive intraductal carcinoma, sporadic; breast cancer, susceptibility to breast cancer, breast cancer type 4, 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's lymphoma, B-cell, Burkitt's, cutaneous T-cell, histiocytic, lymphoblastic, T-cell, thymic, B-cell non-Hodgkin's lymphoma), glioma, adenocarcinoma, adrenal tumor, hereditary adrenal cortical carcinoma, malignant tumors (tumors) of the brain, various other carcinomas (e.g. , bronchogenic large cell, ductal, malignant ascites, Ehrlich-Lettlet ascites, epidermoid, large cell, Lewis lung, medullary, mucoepidermoid, oat cell, small cell, spindle cell, spinous cell, transitional cell, undifferentiated, carcinosarcoma, choriocarcinoma, cystadenocarcinoma), ependymoblastoma, epithelioma, erythroleukemia (e.g., Friend, lymphoblastic), fibrosarcoma, giant cell tumor, glial tumor, glioblastoma (e.g., pleomorphic, astrocytoma), hepatocellular glioma (glioma hepatoma), heterohybridoma, heteromyeloma, histiocytoma, hybridoma (e.g., B cell), adrenal tumor, insulinoma, pancreatic islet tumor, keratoma, epithelioid leiomyoma, leiomyosarcoma, leukemia (e.g., acute lymphocytic, acute lymphoblastic, acute lymphoblastic pre-B cell, acute lymphoblastic T cell leukemia, acute megakaryoblastic, monocytic, acute myeloid, acute myeloid with eosinophilia, B cell, basophilic, chronic myeloid, chronic, B cell, eosinophilic, friend, granulocytic or myeloid, hairy cell, lymphocytic, megakaryoblastic, monocytic, monocytic-macrophage, myeloblastic, myelogenous, myelomonocytic, plasma cell, pre-B cell, promyelocytic, subacute, T cell, lymphoid neoplasm, predisposition to myeloid malignancies, acute non-lymphocytic leukemia), lymphosarcoma, melanoma, breast tumor, mast cell tumor, medulloblastoma, mesothelioma, metastatic tumor, monocytic tumor, multiple myeloma, myelodysplastic syndrome, myeloma, nephroblastoma, neural tissue glial tumor, neural tissue neuronal tumor, schwannoma, neuroblastoma, oligodendroglioma, osteochondroma, bone myeloma, osteosarcoma (e.g. Ewing), osteoporosis, bone metastasis, papilloma, transitional cell, pheochromocytoma, pituitary tumor (invasive), plasmacytoma, retinoblastoma, rhabdomyosarcoma, sarcoma (e.g. Ewing, histiocytic cell, Jensen, osteogenic, reticular cell), schwannoma, subcutaneous tumor, teratocarcinoma (e.g. pluripotent), teratomas, testicular tumors, thymoma and trichoepithelioma, gastric cancer, fibrosarcoma, glioblastoma multiforme;Any solid or non-solid cancer and / or cancer metastasis, including, but not limited to, Turcot's syndrome with multiple glomus tumors, Li-Fraumeni syndrome, liposarcoma, Lynch cancer familial syndrome type II, male germ cell tumors, mast cell leukemia, medullary thyroid, multiple meningiomas, endocrine neoplasia, myxosarcoma, paraganglioma, familial achromophilic, pilomatrixoma, papillary, familial and sporadic, rhabdoid predisposition syndrome, familial, rhabdoid tumor, soft tissue sarcoma, or glioblastoma;

[0121] In some embodiments, the therapeutic biologic is an immunotherapy. In some embodiments, the immunotherapy is an anti-CD20 antibody. In certain embodiments, the anti-CD20 antibody is rituximab. Certain embodiments of the compositions and methods described herein may be useful for treating non-Hodgkin's lymphoma (NHL) in a subject in need thereof, comprising administering to the subject a pharma- tically effective amount of a composition comprising a plurality of particles suspended in a pharma- tically acceptable liquid carrier, the particles (e.g., substantially all of the particles) comprising at least one therapeutic biologic or salt thereof, and an aggregating agent, wherein the concentration of the aggregating agent in the composition is less than about 50 mg / mL, and the concentration of the therapeutic biologic or salt thereof in the composition is greater than about 250 mg / mL. As described herein, any antibody capable of binding to the CD20 antigen may be used in the methods of the present disclosure. Antibodies that bind to the CD20 antigen include, for example, C2B8 (rituximab; RITUXAN®) (U.S. Pat. No. 5,736,137, expressly incorporated herein by reference), an yttrium-

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

[0122] In certain embodiments of the present disclosure, the anti-CD20 antibody is rituximab. Rituximab is a genetically engineered chimeric mouse / human monoclonal antibody. Rituximab is an IgG, kappa immunoglobulin that contains mouse light and heavy chain variable region sequences and human constant region sequences. Rituximab has a binding affinity for the CD20 antigen of approximately 8.0 nM and is commercially available, for example, from Genentech (South San Francisco, CA).

[0123] In other embodiments, the antibody is trastuzumab (Herceptin; Genentech, San Francisco, Calif.). Trastuzumab is a humanized monoclonal antibody that binds to the extracellular portion of the HER2 ectodomain and prevents dimerization and the cascade that leads to growth factor expression. In certain embodiments, trastuzumab is used to treat new or metastatic cancers of the breast, esophageal, gastric, or other HER2-overexpressing cancers.

[0124] Also provided herein is a method for treating an inflammatory disease in a subject in need of such treatment, comprising administering to the subject a pharma- tically effective amount of a composition comprising a plurality of particles suspended in a pharma- tically acceptable liquid carrier, wherein the particles (e.g., substantially all of the particles) comprise at least one therapeutic biologic or salt thereof, and a flocculating agent, wherein the concentration of the flocculating agent in the composition is less than about 50 mg / mL, and the concentration of the therapeutic biologic or salt thereof in the composition is greater than about 250 mg / mL.

[0125] In some embodiments of the methods described herein, the inflammatory disease includes, but is not limited to, arthritic disease, ophthalmologic disease, retinal disease, Crohn's disease, irritable bowel syndrome, Sjogren's disease, tissue graft rejection, asthma, multiple sclerosis, scleroderma, Goodpasture's syndrome, atherosclerosis, chronic idiopathic thrombocytopenic purpura, Addison's disease, Parkinson's disease, Alzheimer's disease, diabetes, 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.

[0126] In some embodiments, the compositions and methods described herein are useful in treating immune disorders, such as acquired hypogammaglobulinemia secondary to hematological malignancies, chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, inflammatory myopathies, Lambert-Eaton myasthenic syndrome, multifocal motor neuropathy, myasthenia gravis, Mersch-Wortmann syndrome, secondary hypogammaglobulinemia specific antibody deficiency, acute disseminated encephalomyelitis, autoimmune hemolytic anemia; cicatricial pemphigoid. The immune system is useful for treating acne, Evans syndrome, fetal-maternal / neonatal alloimmune thrombocytopenia (FMAIT / NAIT), hemophagocytic syndrome, high-risk allogeneic hematopoietic stem cell transplant, IgM paraproteinemic neuropathy, kidney transplant, multiple sclerosis, opsoclonus-myoclonus-ataxia, post-transfusion purpura, toxic epidermal necrolysis / Stevens-Johnson syndrome (TEN / SJS), toxic shock syndrome, Alzheimer's disease, multiple myeloma, sepsis; B cell neoplasms, trauma, or bacterial, viral, or fungal infection. In some embodiments, the immune system disorder is an autoimmune disorder. In certain embodiments, autoimmune diseases include, but are not limited to, multiple sclerosis, scleroderma, type I diabetes, rheumatoid arthritis, thyroiditis, Raynaud's syndrome, Sjogren'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. "Reduce" or "reduce" means to become less or smaller in number, amount, size, or intensity. In certain embodiments, reducing the risk of a disease (such as, for example, focal segmental glomerulosclerosis (FSGS)) comprises 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 comprises delaying the onset of the disease, e.g., by at least about 6 months, e.g., by about 1 year, e.g., by about 2 years, about 5 years, or about 10 years.

[0127] As described herein, particle-based compositions with aggregating agents can directly address the current challenges of subcutaneous administration of therapeutic biologics, e.g., mAbs, by enabling injectability at low injection forces without the need for resuspension prior to injection. In some embodiments, the particles and aggregating agents 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, pre-filled portable drug delivery injection devices, or pre-filled orally administered liquid syringe capsules. The highly dispersible nature of the particles in the compositions described herein allows for patient-friendly subcutaneous injection. Within the subcutaneous space, the therapeutic biologic contained in the particles, as described herein, readily reverts to its original monomeric state upon injection, allowing for full bioavailability. In certain embodiments, the composition does not compromise the quality of the therapeutic biologic, achieves higher loadings, and thus allows the therapeutic biologic, e.g., mAb, to be easily delivered by subcutaneous injection to treat a disease or condition in a subject in need thereof, wherein the disease or condition is cancer, an inflammatory disease, or an immune disease. 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.

[0128] The pharmaceutical compositions and methods disclosed herein can be administered to a subject by any suitable route of administration, including, for example, parenterally or intraperitoneally. In some embodiments, the compositions are administered by parenteral, subcutaneous, oral, epidermal, intradermal, intramuscular, intraarterial, intraperitoneal, or intravenous injection. In certain embodiments, the compositions are administered by subcutaneous injection. Details of suitable routes of administration and compositions suitable therefor can be found, for example, in U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970, and 4,172,896, and patents cited therein. The term "pharmaceutical composition" as disclosed herein refers to a preparation that is in a form that renders the therapeutic biological agent in the composition effective when administered to a subject, and does not contain additional components that are unacceptably toxic to the subject to which the composition is administered. Such compositions are sterile. A "sterile" composition or formulation is aseptic or free of all living microorganisms and their spores.

[0129] The term "injectability" or "syringability" refers to the relative ease with which a liquid composition described herein can be administered to a subject through the use of an injection device, such as a syringe, a portable drug delivery injection device, or an orally administered liquid injector capsule. In particular, injectability is affected in part by the viscosity of the composition, and more importantly, the settling of particles, the injection or transfer flow rate, and the characteristics of the needle, such as 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 breakaway and / or sliding forces required to actuate an injection device consisting of a barrel, a plunger, and a needle. In certain embodiments, the barrel of the syringe has an internal diameter of at least 6 mm. As described herein, the injectability of a composition comprising a plurality of particles comprising a flocculant and at least one therapeutic biological agent is superior to that of an aqueous composition having about the same concentration of an aqueous monomeric therapeutic biological agent. The term "injectability" or "syringability" can also refer to the ability to inject 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 long. Injectability depends on factors such as the pressure or force required for injection, uniformity of flow, quality of aspiration, and lack 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 particle composition without the addition of an aggregating agent. The compositions described herein can improve injectability after a period of time, where the injection force is reduced by at least 10%, preferably at least 30%, more preferably at least 50%, and most preferably at least 75% when compared to a standard particle composition with the same concentration of therapeutic biological agent under otherwise the same conditions.Alternatively, the injectability of a composition can be evaluated by comparing the time or injection force required to inject a flocculated volume, for example, 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 a syringe is pressed with the same force over a period of time, for example, with sedimentation of particles over a period of time. The phrase "flow rate" refers to the volume of liquid composition that can pass through a given cross-sectional area per unit time. In general, 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 / sec.

[0130] The term "injection break-off force" refers to the force required to overcome the friction between the syringe barrel and plunger of a standard injection device before ejection of the syringe contents can occur at a steady rate, e.g., the maximum force required to break the static friction of the plunger. The force is applied at the outward-facing end of the syringe plunger shaft and is directed along the axis of the syringe barrel. The syringe contents are ejected through a syringe needle of a given gauge and length. In certain embodiments, the injection break-off force is measured by a load cell located at the outward-facing end of the syringe plunger during actuation.

[0131] The terms "syringe force", "injection force", "injection glide force", or "glide force" are used interchangeably herein and refer to the force required to maintain steady discharge of the contents of a standard injection device, e.g., the force required to maintain the movement of the plunger once static friction has been overcome. The force is applied at the outwardly facing end of the syringe plunger shaft and is directed along the axis of the syringe barrel. The contents of the syringe are discharged through a syringe needle of a given gauge and length. The term "Newtonian region" or "N" refers to the range of shear stress that is linearly or nearly linearly proportional to the local strain rate at all points. In some embodiments, the addition of a flocculant to a suspension of particles reduces the syringe force (see, e.g., Examples 16 and 17).

[0132] Administration of the compositions described herein may include administration using an 18-33 gauge needle. The 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, administration of the composition uses a 27-33 gauge needle. In some embodiments, the 27-33 gauge needle has a length of about 13 mm (1 / 2 inch) or less. In certain embodiments, the composition is dispensed from a needle having a gauge in the range of 18 gauge to 33 gauge, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 gauge to 33 gauge. In some embodiments, the composition is dispensed from a needle having a gauge in the range of 27 gauge to 30 gauge. In certain embodiments, the composition is dispensed from a needle having a gauge in the range of 25 gauge to 27 gauge. In certain embodiments, the composition is dispensed from a needle having a gauge of 27 gauge.

[0133] In some embodiments, the composition is dispensed 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 dispensed using an injection force of less than about 25 N. In certain embodiments, the composition is dispensed using an injection force of less than about 20 N. In certain embodiments, the composition is dispensed using an injection force of less than about 15 N. In some embodiments, the composition is dispensed using an injection force of less than about 10 N. In certain embodiments, the composition is dispensed using an injection force of less than about 5 N. In some embodiments, the injection force increases at a lower rate than the viscosity of the composition as the concentration of the therapeutic biologic 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.

[0134] In other cases, the compositions comprising a plurality of particles and a flocculating agent described herein optionally further comprise administering at least one hyaluronan degrading agent, e.g., hyaluronidase, administered simultaneously, sequentially, or intermittently with the composition. In some embodiments, the composition administered is less than about 20.0 mL, e.g., 15.0, 10.0, 5.0, 2.0, 1.5, 1.0, 0.5 mL. In some embodiments, the composition administered is less than about 2.0 mL. In certain embodiments, the composition administered is less than about 1.5 mL. In some embodiments, the composition administered is less than about 1.0 mL. In certain embodiments, the composition administered is less than about 0.5 mL.

[0135] In some embodiments, the composition dissolves in less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 minutes after administration. In certain embodiments, the composition dissolves in less than about 60 seconds after administration, e.g., 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, ... 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 seconds. In certain embodiments, the composition dissolves immediately after administration.

[0136] The compositions described herein can be used for parenteral administration, for example, formulated for injection via intravenous, intramuscular, subcutaneous, or even intraperitoneal routes, without the need for manual stirring or premixing of the injection device before use. Pharmaceutical compositions suitable for injectable use include sterile non-aqueous liquid carriers that contain sterile particles. In all cases, the composition must be sterile and must be fluid to the extent that it can be easily injected. Pharmaceutical compositions should also be stable under current good manufacturing practice (cGMP) and storage conditions.

[0137] The present disclosure generally relates to compositions for use in a syringe or portable drug delivery injection device. In certain embodiments, the composition is in a syringe or portable drug delivery injection device. For example, the syringe disclosed herein can be a pre-filled syringe. 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 injectors, automatic injectors (e.g., automatic injection devices, patches), or orally administered liquid injection capsules (e.g., robotic pills that deliver drugs to the gastrointestinal tract, etc.). The compositions described herein can be enclosed in disposable syringes, cartridges, or any other container closures made of glass or plastic, etc. 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 drugs to a patient using a wireless communication system.

[0138] Autoinjectors, such as autoinjectors, pen injectors, or medicine pens, or orally administered liquid syringe capsules, are pre-filled, pre-assembled injection devices that allow parenteral administration of therapeutic agents. The advantage of autoinjectors is that they contain a measured dose of therapeutic agent in a sealed, sterile cartridge or injection device. Autoinjectors are useful for people with poor dexterity, poor eyesight, or who need portability to administer therapeutic agents on time, and can also reduce fear or difficulty of self-injecting therapeutic agents, which increases the likelihood that a person will take the medicine. Autoinjectors are most useful in emergency situations that allow for quick and simple self-administration of therapeutic agents without the need to measure the dosage.

[0139] Ensuring complete delivery of particle suspensions of therapeutic biological agents in the compositions and methods disclosed herein is often important for particle suspensions where injection forces 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 settling in the composition, making user operation difficult.

[0140] In autoinjectors, particles of the therapeutic biological agent in the composition are likely stored as a particle suspension that is then injected. However, long-term storage of particle suspensions (e.g., 3-12 months) has drawbacks such as particle settling that can lead to high injection forces. These autoinjectors require the user to manually shake the syringe body to promote resuspension of the particles immediately prior to injection. Unfortunately, steps such as manually shaking the autoinjector increase the time required to administer a dose of the therapeutic agent, which is undesirable in many emergency medical situations where rapid delivery of the therapeutic agent is required.

[0141] In some embodiments, the composition is administered by a syringe or a portable drug delivery injection device. In certain embodiments, the composition is in a syringe or a portable drug delivery injection device. In certain embodiments, the composition is administered by a syringe. In certain embodiments, the composition is administered by a portable drug delivery injection device. In certain embodiments, the portable drug delivery injection device is a pen injector. In some embodiments, the portable drug delivery injection device is an autoinjector. In certain embodiments, the portable drug delivery injection device is an orally administered liquid injection capsule. 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.

[0142] In some embodiments, the composition is administered by a syringe. In some embodiments, the composition is administered by a pen injector. In certain embodiments, the composition is administered by an auto-injector. In some embodiments, the composition is administered by an orally administered liquid injector capsule. In certain embodiments, the syringe, pen injector, auto-injector, or orally administered liquid injector capsule is pre-filled.

[0143] The compositions of the present disclosure may be utilized to treat a disease or condition in a subject in need of such treatment. 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., a human or an animal. The compositions described herein can be administered to a mammal, e.g., a human or an animal subject, in vivo using a variety of known routes and techniques. For example, the compositions may be provided as an injectable suspension and administered via parenteral, subcutaneous, oral, epidermal, intradermal, intramuscular, intraarterial, intraperitoneal, or intravenous injection using a conventional needle and syringe or a handheld drug delivery injection device. In certain embodiments, the compositions are administered to a mammal. In certain embodiments, the compositions are administered to a human.

[0144] In certain embodiments according to the present disclosure, when the composition is administered, complete dissolution can occur immediately or within a few seconds, reducing any immunological risk posed by particles persisting in the subcutaneous space. For example, compared to an equivalent dose of aqueous monomeric mAb, dosing of particles containing mAb has been shown to produce similar or improved pharmacokinetic (PK) profiles (AUC, Cmax and Tmax) as described herein. In certain embodiments, the composition dissolves immediately after administration.

[0145] The compositions described herein demonstrate subcutaneous delivery of high concentrations of therapeutic biologics (300-750 mg / mL) compositions without loss of biological activity. This has been achieved for a variety of therapeutic biologics by using organic solvents, e.g., sesame oil, medium chain triglycerides (MCT), propylene glycol diesters of saturated vegetable fatty acids C8 and C10 (PGD), and fatty acid esters (ethyl oleate (EO)) as liquid carriers. For example, ethyl oleate is a fatty acid ester with a viscosity of approximately 6 mPa·s at 25°C and does not chemically interact with particles during storage or dissolution in vivo.

[0146] Dense (1.32 g / cm) with controllable size distribution (polydispersity index <0.2) 3 The generation of round particles of 0.01 mm to 0.1 mm diameter can be achieved using various therapeutic biologics described herein. Focused ion beam scanning electron microscopy (FIB-SEM) can be used to determine whether the particles contain no void space, which is crucial to reaching high protein loading 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 can be about 10 to about 80 μm, which is preferred to reach a low viscosity composition, but small enough to prevent syringe clogging in a 27 gauge needle. For example, by loading a pre-filled syringe with flocculating agent and particles at a protein concentration of about 400 mg / mL in an ethyl oleate (EO) liquid carrier, a therapeutic composition can be formed with a viscosity of about 20 mPa·s (correlating to an injection force of about 4 N), which can be stored for at least one month without substantial change in flocculation volume, viscosity, or injection force (e.g., the particles remain substantially suspended in the liquid carrier for at least one month). See Examples 5 and 6. In some embodiments, the composition has substantially the same aggregate volume for at least one month. See Example 6. In some embodiments, the plurality of particles and the aggregate agent remain substantially suspended in the liquid carrier for at least three months. See Examples 10 and 11. In certain embodiments, the injection force remains substantially the same for at least three months under container-closed storage conditions at about 25° C. See Examples 10 and 11. In some embodiments, the yield stress remains substantially the same for at least one month under container-closed storage conditions at about 25° C. See Example 13. In certain embodiments, the addition of the aggregate agent to the composition prevents the particles from settling and prevents clogging of the needle.

[0147] As described herein, the structural stability of particles in the composition is characterized by size exclusion chromatography (SEC), differential scanning fluorimetry (DSF), circular dichroism (CD), cation exchange chromatography (CIEX) and sub-visible particle (SvP) analysis.In addition, pharmacokinetics (PK) and immunogenicity studies are used to analyze the preservation of biological activity.See Examples 7 and 8.

[0148] Also described herein is that SEC data confirmed that minimal aggregate formation was observed during processing compared to the reformulated particle composition that contained 96.6% monomer (protein content 507 mg / mL) compared to the labeled formulation (aqueous mAb as FDA approved formulation) that contained aggregates. See Example 11. After 30 days of storage at 40°C, DSF showed less than 1°C thermal shift across the samples, and CD could not detect any difference in secondary structure (beta-sheet percentage). CIEX was used to analyze the charge variants of the protein as required by regulations (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 due to the protein being more stable in the solid state as particles.

[0149] After 30 days storage at 40°C, flow cytometry assay shows the preservation of biological activity.No discernible difference was found between FDA-labeled formulation and those compositions described herein.As shown in Example 4 and Example 5 herein, in the case of FDA-labeled formulation, biological activity significantly decreased after storage, but no decrease in activity was observed for particles in the composition.

[0150] Prior to the initiation of in vivo studies, the particles used in the compositions were obtained under substantially sterile conditions. Bacterial endotoxin levels were approximately three orders of magnitude below accepted injection standards (0.05EU / mg to 0.25EU / mg). In addition, microbial growth assays showed no observable growth.

[0151] The compositions described herein are comparable to aqueous FDA-labeled formulations in terms of rat pharmacokinetics (PK), SC clearance (mouse), and efficacy (mouse xenografts). The PK profile is shown in Example 7, where the mAb microparticle suspension (SC injection) shows higher bioavailability than the aqueous mAb SC injection. See FIG. 11. The in vivo dissolution behavior of the particles has been shown to ensure that the composition can clear the injection site at an increased rate compared to standard aqueous formulations, as undissolved particles can potentially cause immunogenic reactions. In some embodiments, the compositions have improved pharmacokinetics (PK) compared to aqueous compositions that contain at least one therapeutic biologic in soluble form. See FIG. 11. The area under the curve (AUC) from time zero to infinity represents the total drug exposure over time. Peak concentration is the pharmacokinetic measure used to determine drug dosing. Maximum concentration (Cmax) is the highest concentration of drug in blood, cerebrospinal fluid, or target organ after a dose is given. Tmax is the time it takes for a drug to reach its maximum concentration (Cmax) after administration of a drug that needs to be absorbed (e.g., an oral drug). Tmax is governed by the rate of drug absorption and the rate of drug elimination, which are equal at Tmax. In certain embodiments, the composition has improved AUC, Cmax, and / or Tmax compared to an aqueous composition comprising at least one therapeutic biologic in soluble form.

[0152] In some embodiments, administration of hyaluronidase allows for an increase in the volume and fluid dispersion of the composition with the flocculant administered by subcutaneous syringe injection. As disclosed herein, the injection volume of the composition administered subcutaneously without resuspension can be significantly increased by pre-injection with hyaluronidase. As shown in Example 9, these hyaluronidase pre-injection sites showed improved speed of injection of the composition as determined by the appearance of skin blisters formed at the site of administration upon injection.

[0153] Certain embodiments of the present disclosure are described herein. Of course, variations, modifications, alterations, and replacements of equivalents of these specific embodiments will be apparent to those skilled in the art upon reading the foregoing description. The inventors expect that those skilled in the art will use such variations, modifications, alterations, and replacements of equivalents as appropriate, and the inventors intend for the present disclosure to be carried out in other ways than those specifically described herein. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed, altered, or modified to produce essentially similar results. Thus, the present disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the elements described above in all possible variations thereof is encompassed by the present disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

[0154] The present disclosure having been generally described will be more readily understood by reference to the following examples, which are included solely for the purpose of illustrating certain aspects and embodiments of the present disclosure and are not intended to be limiting.

[0155] kit In various embodiments, a kit is contemplated that includes a pharma- ceutically effective composition comprising a plurality of therapeutic particles having an aggregating agent suspended in a pharma- ceutically acceptable liquid carrier. For example, the kit may include one or more additional containers, each having one or more of a variety of materials (e.g., reagents and / or devices, optionally in concentrated form) that are desirable from a commercial and user standpoint for the 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, vials, and / or tube labels listing contents and / or instructions for use, and package inserts with instructions for use. In some embodiments, the present embodiments contemplate a kit for administering the therapy of the embodiments. The kit may include one or more sealed pre-filled syringes, cartridges, or portable drug delivery injection devices containing any of the pharmaceutical compositions of the present disclosure. The kit may include, for example, at least a plurality of particles comprising at least one therapeutic biological agent, as well as reagents for preparing, formulating, and / or administering the components of the embodiments, or for carrying out one or more steps of the disclosed therapeutic methods. In some embodiments, the kit may also include a suitable container that does not react with the components of the kit, e.g., an Eppendorf tube, a syringe, a bottle, a tube, or a portable drug delivery injection device. The container may be made of a sterilizable material, e.g., plastic or glass. In certain embodiments, the composition is dispensed from a pre-filled syringe or a portable drug delivery injection device. In certain embodiments, the kit includes a syringe or a portable drug delivery injection device, and a composition comprising a plurality of particles suspended in a pharma- ceutically acceptable liquid carrier, the particles (e.g., substantially all of the particles) comprising at least one therapeutic biologic or a salt thereof, and a flocculating agent, wherein the concentration of the flocculating agent in the composition is less than about 50 mg / mL and the concentration of the therapeutic biologic or a salt thereof in the composition is greater than about 250 mg / mL.

[0156] The kit may further include an instruction sheet outlining the procedural steps of the methods described herein, following substantially the same procedures as those described herein or known to those of skill in the art. The instructional information may be in a computer readable medium containing computer readable instructions that, when executed using a computer, result in a display of an actual or hypothetical procedure for delivering a pharma- ceutical effective amount of a therapeutic biologic.

[0157] A label is optionally on or associated with a container. For example, a label is on a container when letters, numbers, or other symbols forming the label are affixed, stamped, or etched on the container itself, and a label is associated with a container when present in a receptacle or carrier that also holds the container, for example, as a package insert. In addition, a label can be used to indicate that the contents are to be used for a particular therapeutic application. In addition, a label can indicate instructions for using the contents, for example, in the methods described herein. In certain embodiments, the pharmaceutical composition is present in a pack or dispenser device that contains one or more unit dosage forms containing the therapeutic biological agent provided herein. For example, the pack contains a metal or plastic foil, such as a blister pack. Alternatively, the pack or dispenser device can be accompanied by instructions for administration. Alternatively, the pack or dispenser can be accompanied by a notice associated with the container, in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, which notice reflects the agency's approval of the drug form for human or veterinary administration. Such notice, for example, is the labeling approved by the U.S. Food and Drug Administration for prescription drugs or an approved product insert. In some embodiments, compositions containing the therapeutic particles and flocculating agents provided herein formulated in a compatible pharmaceutical liquid carrier are prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

[0158] 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, e.g., the composition, is formulated with an administration 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 administration volume of the composition is less than about 2.5 mL. In certain embodiments, the administration 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.

[0159] In other cases, the method further comprises administering at least one hyaluronan degrading agent, e.g., a pharma- tically effective amount of at least one hyaluronan degrading agent administered simultaneously, sequentially, or intermittently with the composition. In some embodiments, the hyaluronan degrading agent administered is in a volume of less than about 2.0 L, e.g., less than about 1.8, 1.5, 1.2, 1.0, 0.8, 0.5, 0.3, 0.1 L, or less than about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9.0, 8.0, 7.0, 6.0, 5.0, 4.0, 3.0, or about 2.5 mL. In some embodiments, the composition administered is less than about 20.0 mL. In certain embodiments, the composition administered is less than about 10.0 mL. In some embodiments, the composition administered is less than about 5.0 mL.

[0160] In some embodiments, such kits include a pre-filled injection device of the present disclosure, such as a syringe or a portable drug delivery injection device, in a blister pack. The blister pack itself may be internally sterile. In some embodiments, a pre-filled injection device according to the present disclosure, such as a syringe or a portable drug delivery injection device, may be placed inside such a blister pack before undergoing sterilization, such as final sterilization.

[0161] The present disclosure having been generally described will be more readily understood by reference to the following examples, which are included solely for the purpose of illustrating certain aspects and embodiments of the present disclosure and are not intended to be limiting.

[0162] Example [Table 1-1] [Table 1-2] [Table 1-3]

[0163] material Human IgG (IRHUGGF-LY, >97%) and bovine IgG (IRBVGGF) were obtained from Innovative Research as powders or as aqueous solutions. Bovine serum albumin (BSA) and human serum albumin (HSA) were purchased from Sigma-Aldrich. Monoclonal antibodies (mAbs) were provided and received as aqueous solutions. Roche Rituximab biosimilar 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. Roche Trastuzumab biosimilar was purchased from Genscript Biotech Corporation that provided the antibody as an aqueous composition. Custom "feed solution" compositions used to process the particles were generated by modifying the received formulations by desalting followed by concentration and addition of desired excipients or by direct buffer exchange. All excipients used in the particle compositions are used in existing approved biologics injections. Concentration columns were sourced from Millipore Sigma (Amicon® Ultra 15 mL filters for protein purification and concentration with 10-50 kDa cutoff) and were used as needed (i) to reach the desired monoclonal antibody concentration and (ii) to exchange buffers / excipients prior to particle formation. In certain cases, Zeba desalting columns (THERMO FISHER SCIENTIFIC™ 87773) were also used to remove salts from the solution. Typically, the ratio of residual salt to protein (wt / wt) in the desalted solution was less than 1%, as determined form conductivity measurements and / or elemental analysis. All excipients were purchased from Sigma-Aldrich and used as received.

[0164] method FLOWCAM™: Particle sizing was measured using FLOWCAM™, a dynamic image analysis instrument. Samples were 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 weighted).

[0165] Image analysis: Particle diameter and circularity were measured using ImageJ analysis on SEM images. Analysis was performed on images at 500x or 1000x, for example. The ImageJ particle analysis tool was run on the images to identify objects with a circularity of >0.8 and a size of >0.5 μm, each accompanied by an object outline. These outlines were visually inspected for a good fit. Any incorrectly identified particles were manually rejected and any missing particles were manually included and measured using the ImageJ diameter tool. Selected microscopy images were chosen for further analysis based on (i) minimal particle overlap, (ii) good contrast between particles and background, and (iii) a resolution that provided a 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. The ImageJ tool "Analyze Particles" was then applied on the binarized pictures with the following parameters: circularity between 0.5 and 1.0; size between 5 and infinity square microns; exclusion on edges; filling of holes. The contours of the identified particles were then superimposed on the original image. Incorrectly identified particles were then discarded, e.g. single particles whose contours did not match any particle or clusters identified as particles. Missing particles were measured by manually tracing the particle contours and using the measurement tools of ImageJ.

[0166] Accelerated Storage Protocol: All samples were transferred to glass bottom plates and 2R Schott vials for aging (typically 2 mL or 4 mL 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 over the aging period to ensure integrity and stability.

[0167] Viscosity measurements: Unless otherwise stated, suspension viscosity was measured at 25°C using an AR-G2 rheometer (TA Instruments) and a 25mm plate. Measurements were performed at 1000s-1, which is below the shear rate experienced by a 27 gauge needle. Each measurement was repeated three times (with an interval of approximately 60 seconds between repeats) to assess the short-term physical stability of the suspension. Calibration standards were recorded and instrument settings were verified before each measurement.

[0168] Injectability measurements: Unless otherwise stated, syringe forces (injection forces) were measured during 0.1 mL / sec ejection of 1 mL of suspension (400 mg / mL particles) using a custom-made force sensor device and a 1 mL Norm-ject model syringe with a 27 gauge ultra-thin wall needle (TSK).

[0169] Karl Fischer: Tests for moisture content were performed using Karl Fischer analysis: Approximately 100 mg of particles were heated to 150° C. in an oven and the released water was determined coulometrically.

[0170] Skeletal density: Skeletal density was measured by gas pycnometry. The gas was nitrogen or other compatible gas and the particle mass was 0.0413 g.

[0171] Particle dissolution: Phosphate-buffered saline (PBS) was added to the dried particle samples to produce a final concentration of 50 mg / mL (particle mass / mL of solution, 96-well plate). After a period of time, a 10 μL aliquot was removed 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.

[0172] Salt content: Salt content was recorded by measuring sodium content using inductively coupled plasma optical emission spectroscopy (ICP-OES). A calibration curve was prepared using a sodium standard (ICPTRACECERT®, 1000 mg / L). Quality control was completed using a standard solution diluted with 100 ppm sodium. A sample of particles (approximately 15 mg) dissolved in 2% by volume nitric acid (10 mL) was then analyzed, resulting in an intensity below the instrument detection limit of approximately 0.5 ppm for sodium. This indicated a sodium content of less than 0.034% by weight, and a total salt content of less than 0.1% by weight (assuming that sodium citrate and sodium chloride are equally removed).

[0173] Size Exclusion Chromatography (SEC) Measurement: A 20 μL injection of sample (1 mg / mL) was performed on an AGILENT ADVANCEBIO™ SEC (300 mm×2.7 μm, 300 Å column) in SEC buffer (25 mM phosphate, 250 mM NaCl pH 6.8) at a flow rate of 1 mL / min for 15 min. Peak analysis was performed by auto-integration using the following parameters: gradient sensitivity=0.5, peak width=0, height reject=0, area reject=0, shoulder off, area percent reject 0, standard tangent skim mode, advanced baseline correction, 0 for front peak skim height ratio, 0 for tail peak skim height ratio, 0 for peak to valley ratio, and 0 for skim valley ratio. Alternatively, 20 μL injections of sample (1 mg / mL) were performed on an AGILENT ADVANCEBIO™ SEC (300 mm×2.7 μm, 300 Å column) in SEC buffer (25 mM phosphate, 250 mM NaCl pH 6.8) at a flow rate of 1 mL / min for 15 min. Peak analysis was performed by auto-integration using the following parameters: gradient sensitivity=0.5, peak width=0, height reject=0, area reject=0, shoulder off, area percent reject 0, standard tangent skim mode, advanced baseline correction, 0 for front peak skim height ratio, 0 for tail peak skim height ratio, 0 for peak to valley ratio, and 0 for skim valley ratio.

[0174] Cation Exchange Chromatography (CIEX) Measurements: Charge variant analysis was performed on each sample under accelerated storage conditions at 0, 7, and 30 days 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 with Buffer A: 30 mM phosphate, pH 6.3, and NaCl: 175 mM. Samples were run in a gradient set to start with 100% Buffer A and ramp to 100% Buffer B over a period of 20 minutes, then ramp back to 100% Buffer A and 0% Buffer B in the next minute. The system was re-equilibrated in 100% Buffer A for 10 minutes before the next sample was injected. Integration was performed as manual skim peak mode and reflected AGILENT™ data.

[0175] Flow cytometry: One 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 the well at a starting concentration of 200 μL. The dilution factor for mAb labels, particles, and suspensions was 3X. The plate was incubated at 4° C. for 30 minutes. The plate was 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 secondary antibody at a 1:200 dilution. The plate was 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.

[0176] Scanning Electron Microscopy (SEM): Electron micrographs were collected on selected samples on either a HITACHI™ TM3030Plus or TM1000 tabletop microscope. Samples were immobilized on conductive tape and examined in a low vacuum antistatic environment, eliminating the need for sample preparation.

[0177] Image analysis: Selected microscopic images were selected for further analysis based on (i) minimal particle overlap, (ii) good contrast between particles and background, and (iii) a resolution that provided 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. The ImageJ tool "Analyze Particles" was then applied on the binarized pictures with the following parameters: circularity between 0.5 and 1.0; size between 5 and infinity square microns; exclusion on edges; filling of holes. The outlines of the identified particles were then superimposed on the original image. Incorrectly identified particles were then discarded, e.g., single particles whose outlines did not match any particle or clusters identified as particles. Missing particles were measured by manually tracing the particle outline and using the measurement tools in ImageJ.

[0178] Density Analysis: The skeletal density of particles from selected samples was determined by testing approximately 0.1 g of powder with an ACCUPYC™ II 1340 gas displacement pycnometer system.

[0179] Water Content Analysis: The moisture in the particles from selected samples was determined by placing approximately 0.1 g of powder in an oven with a Karl Fischer titrator and heating the sample.

[0180] ELISA assay: ELISA assay was used on selected samples to detect human antibodies in a denaturing sensitive format. Human IgG was first plated in PBS for 1 hour, followed by washing 3 times with washing buffer (PBS+0.05% Tween20) for 4 minutes, followed by blocking with 2% BSA (Sigma) in washing buffer for 45 minutes, followed by incubation with diluted (20 μg / mL) Protein A-HRP (ABCAM™) for 45 minutes, followed by washing 3 times with buffer for 3 minutes, followed by incubation with TMB (ABCAM™) for 10 minutes, and finally followed by quenching the reaction with stop solution (ABCAM™). Colorimetric reading was performed on a THERMO MULTISKAN SPECTRUM™.

[0181] Sub-visible Particle (SvP) Analysis: Sub-visible particles (SvP) were analyzed on a Fluid Imaging Technologies FLOWCAM™ PV-100 system. Samples for analysis were reconstituted to the desired concentration with filtered water (MILLI-Q™) in sterile centrifuge tubes. Three sets of samples were then investigated. 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.

[0182] Accelerated Storage: Unless otherwise stated, storage was performed under accelerated conditions for selected samples by keeping them at elevated temperature (40° C.) in an incubator or oven for a specified period of time. Samples were kept in 2 mL or 4 mL WHEATON™ glass vials and sealed with paraffin film.

[0183] Inverse Gas Chromatography (IGC): Inverse gas chromatography was used to analyze the powdered samples. A cylindrical column was packed with 200-300 mg of powdered sample to create the stationary phase. After an inert gas purge, a series of gas probes were injected onto the column. Determination of the retention volume for each probe allowed the evaluation of the dispersive and polar components of the surface energy for each sample.

[0184] X-ray diffraction (XRD): Samples were packed into 0.7 mm diameter glass capillaries. Powder patterns were measured on a PANALYTICAL EMPYREAN™ diffractometer equipped with an incident beam focusing mirror and an X'CELERATOR™ detector. Patterns (1-50 o 2θ, 0.0167113 o Step, 4 sec / step, 1 / 4 o Divergence slits, 0.02 rad Soller slits) were measured using Mo Ka radiation. When electrostatic effects (which occurred after grinding in a mortar and pestle when evaluating the freeze-dried control) prevented the sample from filling the capillary, the powder patterns were measured from flat-plate specimens on a BRUKER™ D2 Phase diffractometer equipped with a LYNXEYE™ position-sensitive detector. Patterns were recorded from 5 to 100 nm, counting for 1.0 sec / step. o Using Cu Kα radiation at 2θ 0.0202144 o The measurements were performed at standard instrument settings (30 kV, 10 mA, 0.6 mm divergence slit, 2.5 o Soller slits, and a scattering screen height of 3 mm) were used.

[0185] Microflow Particle Sizing (MPS): Flow imaging microscopy for particle size analysis was performed using a FLOWCAM™ PV-100. To investigate the size and dispersity of the particles, 5 mg of powder was dispersed in 10 mL of dry isopropanol via sonication. The isopropanol continuous phase prevented the particles from dissolving, i.e., preventing reconstitution. 0.3 mL was injected into the cell and images of the particles were acquired using a flow rate of 0.15 mL / min. Particles with a circularity greater than 0.9 were reported in the analysis, any duplicate images were removed from the analysis, and the size distribution and dispersity of particles in the range of 1-100 μm were obtained.

[0186] Dynamic Vapor Sorption (DVS): The powders were analyzed using dynamic water vapor sorption. Approximately 50 mg of powdered sample was loaded into the pan of the instrument's microbalance. The sample was held isothermally 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 ramped to 90% RH at a constant rate of 4% RH per hour. The sample was held at 90% RH for 1 hour and then the RH was reduced to 0% as a step change. The sample was held at 0% RH for 1 hour after which the measurement was terminated.

[0187] Dynamic Scanning Calorimetry (DSC): The powdered samples were analyzed using dynamic scanning calorimetry. 5-10 mg chunks of powdered sample were loaded into aluminum crucibles and hermetically sealed. The crucibles were loaded into the instrument and the heat flow to the sample was monitored while the temperature was ramped from -80 to 200°C, optionally from 20 to 180°C at a constant rate of 5°C / min.

[0188] USP <790> :USP <790> According to the standard, the dissolved particle samples were visually observed against white and black backgrounds under lighting conditions of more than 2000 lux. A high density polyethylene sheet with a matte finish was selected for the background to reduce glare. The illuminance at the viewing point was checked with a lux meter (Dr.Meter, LX1330B). After mixing, the samples were held against the background and viewed for 5 seconds.

[0189] Flocculated Volume: Flocculated volume can be measured by suspending particles in a desired liquid carrier at a suitable concentration. The suspension is then agitated, typically by shaking or mechanical means, to ensure uniform dispersion of the particles in the fluid at the start of the measurement. Timing begins immediately after agitation is stopped. The mixture is allowed to settle and the extent of settling or settling of solids is observed and recorded over time.

[0190] Example 1 General protocol for preparation of protein particles: 1. An aqueous protein solution was adjusted to reach a final concentration of 50-160 mg / mL. 2. Excipients were then added to the resulting solution, which was then filtered through a small 0.22 μm filter to remove any extraneous solids from the resulting aqueous "feed" solution. 3. A dehydrating solvent (e.g., n-butyl acetate) was added to the vessel (2 mL-200 L vessel). 4. The protein feed solution was then added to the vessel at various flow rates (e.g., up to 100 mL / min). 5. The mixture was then agitated (e.g., subjected to a shear field or up to 25,000 rpm) for a period of time (e.g., up to 5 minutes) to generate aqueous droplets, which were then dehydrated (for a time dependent on the volume of dehydrating solvent, the rpm of the mixture, and the flow rate) to form protein particles suspended in the mixture. 6. The protein particles were then separated from the liquid mixture (e.g., by filtration, centrifugation, decanting, etc.). 7. The liquid mixture (aqueous liquid and dehydration solvent) was removed. 8. Residual solvents (e.g., aqueous liquid and dehydration solvent) in the protein particles were removed from the protein particles (e.g., vacuum drying, gas drying, gas sparging, extraction solvent, etc.), and then the moisture content in the particles was adjusted (e.g., humid gas, extraction solvent, etc.). 9. A portion of the protein particles was then dissolved in DI water and incubated for complete redissolution for characterization. 10. The solution was then analyzed to measure protein quality (SvP analysis using FLOWCAM™, and soluble aggregates (e.g., SEC), fragmentation (e.g., SEC), charge variant (e.g., ion exchange chromatography (SCEX)) analysis using HPLC-SEC), as well as protein concentration. Protein concentrations ranging from 200 mg / mL to 800 mg / mL have been processed according to the general protocol (particle protein loading of at least 60-93% (w / w)). Particles with protein loading up to 85% showed improved stability compared to the starting aqueous feed solution.

[0191] Preparation of Ab particles in target size and dispersibility range: Rituximab, trastuzumab, human or bovine IgG particles were formed under the general protocol to provide 10 mg of particles for analysis. The mean particle size, D10, D50, and D90 were reported based on count-weighted particle size analysis (FLOWCAM™). The mean particle size, D10, D50, and D90 of the particles were determined to have an average particle size of 5-50 μm and D90<60 μm. The particles had the following particle size statistics: mean diameter=5-50 μm. SEM of the particles for the batch was obtained. Images were acquired at 1000x magnification as shown in Figure 1. Image processing and statistics were performed using ImageJ analysis, count-weighted to provide protein particles with the desired physical characteristics (e.g., circularity, internal void space, protein by weight, stability, and quality, etc.) that were used for suspension studies.

[0192] Determination of aggregation, fragmentation, and charge variant changes in particles by SEC: Dry powder samples were dissolved in ultrapure water to 5% (w / v) and shaken at 60 RPM. After triplicate protein concentrations were determined, samples were diluted to 1 mg / mL in PBS and syringe filtered in HPLC vials. Samples in carrier liquid were resuspended, each sample was immediately centrifuged at 500 RPM, and the resulting carrier liquid supernatant was aspirated (and discarded) without disturbing the settled particles. After adding water to the particles and mixing, samples were shaken at 60 RPM. After initial protein concentration determination (from the bottom or aqueous layer), each sample was diluted with PBS and protein concentration was measured in triplicate (from the bottom or aqueous layer). Each sample was then diluted with PBS and syringe filtered in HPLC vials, discarding the needle before filtration (to prevent transfer of carrier liquid). All samples were analyzed using an AGILENT™ 1260 Infinity II bio-inert LC system and a TSKgel SuperSW HTP column (4 μm, 4.6 mm ID×150 mmL) equilibrated with 200 mM arginine-HCl, 100 mM sodium phosphate pH 6.5. The autosampler and column compartments were maintained at 4° C. and 20° C., respectively, and UV absorbance was monitored at 280 nm. Run time was 10 minutes. Area % of integrated high molecular weight (aggregates), monomer, and low molecular weight peaks were reported.

[0193] Determination of aggregation, fragmentation, and charge variant changes in particles by SCEX: Dry powder samples were dissolved in ultrapure water to 5% (w / v) and shaken at 60 RPM. After triplicate protein concentrations were determined, samples were diluted with ultrapure water and syringe filtered in HPLC vials. Samples in carrier liquid were resuspended, each sample was immediately centrifuged at 500 RPM, and the resulting carrier liquid supernatant was aspirated (and discarded) without disturbing the settled particles. After adding water to the particles and mixing, samples were shaken at 60 RPM. After initial protein concentration determination (from the bottom or aqueous layer), each sample was diluted with ultrapure water and protein concentration was measured in triplicate (from the bottom or aqueous layer). Each sample was then diluted with ultrapure water and syringe filtered in HPLC vials, discarding the needle before filtration (to prevent transfer of carrier liquid). All samples were analyzed using an AGILENT™ 1260 Infinity II bio-inert LC system and a THERMO SCIENTIFIC MABPAC™ SCX-10 RS column (5 μm, 2.1 mm ID x 150 mmL) 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 compartments were maintained at 4°C and 30°C, respectively, and UV absorbance was monitored at 280 nm. The area % of the integrated acidic, neutral (major), and basic peaks was reported.

[0194] Example 2 General protocol for protein particle 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) were made into a suspension at a concentration of 200-700 mg / mL 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 of saturated vegetable fatty acids C8 and C10 (PGD), triacetin, caprylic triglyceride). This was achieved by adding the protein particles to the organic carrier liquid and using a mixer (e.g., rotor-stator, etc.) to generate a diluted suspension. The diluted suspension was sieved through a 60 µm filter and the suspension was adjusted to the desired protein concentration. To this suspension, flocculants were added to reach a total concentration of flocculant up to 10 mg / mL with a protein concentration of 200-800 mg / mL. The suspension was initially mixed using a vortex and / or mechanical mixing for 1 min to create the desired flocculation volume that remained stable for at least 1 month.

[0195] HIgG particle composition: HIgG particles (74% protein:2% histidine:24% arginine:1% PS80 (or PS20), or 78% protein:2% histidine:19% arginine:1% PS80, or 89% protein:3% histidine:7% arginine:1% PS80, or 89% protein:3% histidine:7% arginine:1% trehalose (or methionine), or 88% protein:3% histidine:7% arginine:1% PS80:1% methionine (or NaCl, sucrose, proline, sodium phosphate)) were made into a suspension in ethyl oleate at a concentration of 500 mg / mL. This was accomplished by adding 19-23 g of particles to approximately 450 mL of ethyl oleate and using a rotor-stator mixer to generate a diluted suspension. The diluted suspension was sieved through a 60 μm filter and adjusted to a concentration of 500 mg / mL HIgG by centrifugation. To an aliquot of this suspension, a flocculant (polysorbate 80) was added by pipette to reach a final total concentration of up to 10 mg / mL with a HIgG concentration of 500 mg / mL. This suspension was vortexed and then mechanically mixed for 1 minute to create a stable suspension (flocculated volume) that remained consistent for at least 1 month compared to the particle suspension without the flocculant. HIgG and BIgG were used interchangeably. Protein characterization was accomplished according to Example 1 and showed improved stability compared to the starting aqueous feed solution.

[0196] Rituximab particle composition: Rituximab particles (98% protein:2% histidine, or 80% protein:2% histidine:18% arginine) were made into suspensions at concentrations of 500 and 700 mg / mL in ethyl oleate. This was accomplished by adding the particles to approximately 450 mL of ethyl oleate and using a rotor-stator mixer to generate a dilute suspension. The dilute suspension was sieved through a 60 μm filter and adjusted to a concentration of 500 or 700 mg / mL rituximab by centrifugation. To an aliquot of this suspension, a flocculant (polysorbate 80) was added by pipette to reach a final total concentration of up to 10 mg / mL at 500 or 700 mg / mL rituximab concentrations. The suspension was vortexed and then mechanically mixed for 1 minute to create a stable suspension (flocculated volume) that remained consistent for at least one month compared to particle suspensions without the flocculant. Protein characterization was accomplished according to Example 1 and showed improved stability compared to the starting aqueous feed solution.

[0197] Trastuzumab particle composition: Trastuzumab particles (71% protein: 2% histidine: 26% arginine: 1% PS80) were made into suspensions at concentrations of 500 or 700 mg / mL in ethyl oleate according to the Rituximab protocol. Stable suspensions (aggregate volume) that remained consistent for at least one month were produced according to the Rituximab protocol. Characterization was accomplished according to Example 1 and showed improved stability compared to the starting aqueous feed solution.

[0198] Example 3 Protein particle compositions with high concentration, stable aggregation volume, and low injection force: The units "mPa·s" and "cP" are used interchangeably in the broadest sense herein.

[0199] Rheology: Suspensions of Rituximab, Trastuzumab, Bovine IgG, or Human IgG particles were made with the following characteristics: protein loading >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 the carrier liquid. At concentrations >300 mg / mL, viscosities of approximately 20 cP (mPa·s) were measured. Even higher concentrations can be achieved if lower viscosity carriers are considered.

[0200] Rheology (Days 0, 7, 30 at 40°C): Viscosity of suspensions of particles (rituximab, trastuzumab, bovine IgG, or human IgG) was tracked over time to ensure that it did not vary by more than 5% over 7 and 30 days of accelerated storage. At each time point, the suspension had a viscosity <20 cP (mPa s) with a protein loading >250 mg / mL. Concentration conditions were met based on the preparation of a 280 mg / mL protein suspension. Day 0 samples were measured immediately. Day 7 and Day 30 samples were aged according to the aging procedure outlined above. 1000 s -1 Measurements were taken at a shear rate of 100 / min. On day 0, the viscosity of the suspension was measured at 16.87 cP. On day 7, the viscosity reduced to 16.33 cP. On day 30, the viscosity increased by 29% to 21.83 cP compared to day 0, just above the 20 cP limit. However, it has been estimated that using such small plates (25 mm), small instrument gaps (150 microns), and small sample volumes, the error in the measurement could be as high as 30%.

[0201] Karl Fischer titration: The water content of the solid particles (rituximab, trastuzumab, bovine IgG, or human IgG) was determined to define the constituent composition of the particles using Karl Fischer titration. The water content of the particles was measured, recorded, and determined to be 1-6 wt% for each of rituximab, trastuzumab, bovine IgG, or human IgG.

[0202] 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 with flocculant was vortexed until visually homogenous. 250 uL was pipetted onto the base plate and the sample was measured at a shear rate of 950 1 / s.

[0203] Protein content determination: The particle suspension with 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 to 5% (w / v) in ultrapure water, and shaken at 60 RPM for 30 minutes. After determining the triplicate protein concentrations (from the bottom or aqueous layer for the suspension), the mass percentage of protein in the particles was calculated by dividing the product of the averaged redissolved protein particle concentration and the redissolution volume by the mass of the microparticles. The averaged concentration of the suspension (corrected by the aqueous / formulation carrier liquid dilution factor) was then divided by this mass percentage of protein in the particles to calculate the theoretical mass of the particles in the suspension. The results correspond to the protein content shown in Examples 1 and 2.

[0204] Dissolution: The rate of dissolution was recorded for each suspension at days 0, 7, and 30. Dissolution was recorded at various time points to confirm dissolution of particles within approximately 20 minutes and approximately 60-80 minutes for the suspensions. Particles and suspensions were dissolved in PBS to a final concentration of 10 mg / mL and rocked on a rocker. Dissolution of particles at days 0, 7, and 30 was also stored at 40°C.

[0205] USP <790> : The presence of visible particles was determined in samples with dissolved particles. USP <790> was used to determine whether there were any particles present in the "visible" range (>100 μm). Observation of dissolved particles was used to assess the presence of visible particles. Observations were made briefly (5 seconds) with white and black backgrounds under appropriate lighting. Some potentially small particles were observed, but were difficult to see with the eye, which may be classified as sub-visible particles. Given that these particles may be considered sub-visible, they were investigated further using other methods as described below.

[0206] Sub-visible particle analysis: The sub-visible particulate matter present in the dissolved particles was investigated. Sub-visible particle analysis was performed after dissolution of the particles. This data was compared with other competitive particle formation techniques. SvP analysis was performed by a particle analyzer, where the particle count was adjusted for background signal in the control sample. The sub-visible particle count measured in the samples was lower than any other finished technique, including standard freeze-drying.

[0207] Flocculation volume: BIgG particles were added to 3 mL of ethyl oleate followed by the addition of a flocculant (lecithin or PS80) at 0.1 wt% to 0.01 wt% to form a protein concentration of 500 mg / mL. The suspension was mixed initially and the flocculation volume was calculated and compared to a suspension without flocculant. The effect of flocculants on particle agglomeration and flocculation volume is shown in Figure 2. Lecithin at 0.01 wt% and 0.1 wt% reduces particle agglomeration and flocculation volume. PS80 .01 wt% and 0.1 wt% increases particle agglomeration and flocculation volume compared to suspension without flocculant. In both cases, no reduction in flocculation volume was observed and it remained stable for at least 3 months at 25C.

[0208] Injectability: The force to inject the suspension with the flocculant through the needle was measured and verified that the injection force remained the same after 24 hours without resuspension before injection. As obtained above, a suspension of BIgG particles at a protein concentration of 500 mg / mL in EO and PS80 at 0.1 wt% was filled into a 5 mL syringe with an 18-G needle (3 inches). The injection force was less than 2.0 N and remained the same after 24 hours of storage. For reference, the force to actuate an empty syringe with a syringe needle was compared using a 15 mPa·s standard, and the results showed that the injection forces were all below 2.0 N. These studies showed that the force to inject the composition remained stable for at least one month.

[0209] Example 4 Particle and suspension quality studies: Design: A suspension of particles with flocculant was obtained using a procedure similar to that of Examples 1-3. The stability of the protein particle suspension with flocculant was evaluated over a 3 month period at 5, 25, and 40° C. to determine the stability of the particle suspension composition compared to a protein liquid drug substance (LDS), e.g., compared to the starting aqueous feed solution.

[0210] Composition: Using a procedure similar to that of Examples 1-3, a suspension of particles with flocculant formulated into the particles (600 mg / mL protein and 0.1 mg / mL PS80; 74% protein: 2% histidine: 24% arginine: 1% PS80) was obtained, which was then suspended in ethyl oleate.

[0211] Buffer exchange by tangential flow filtration (TFF): Protein 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.

[0212] FIB-SEM Imaging: Particles were imaged using a scanning electron microscope (HITACHI™, TM-1000). Particle samples were mounted on an adhesive stage for analysis. Images were captured at various magnifications using an accelerating voltage of 15 kV.

[0213] Buffer exchange by tangential flow filtration (TFF): Protein 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.

[0214] SEM Imaging: Particles were imaged using a scanning electron microscope (HITACHI™, TM-1000). Particle samples were mounted on an adhesive stage for analysis. Images were captured at various magnifications using an accelerating voltage of 15 kV.

[0215] Karl Fischer Coulometry: Testing for moisture 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.

[0216] 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 period of time. The final dissolved concentration was recorded by removing an aliquot from the sample and measuring the absorbance at 280 nm (using an extinction coefficient E1%=1.69 L g-1 cm-1).

[0217] Turbidity: An aliquot of the reconstituted particle solution was transferred to a 1-cm path length cuvette. The absorbance at 405 nm was recorded using a NANODROP™ One UV-VIS spectrophotometer (THERMO SCIENTIFIC™). The results showed improved turbidity compared to the starting aqueous feed solution and particles.

[0218] Size Exclusion Chromatography (SEC) Measurement: Sample injections were performed on a Tosoh TSKgel SuperSW mAb HTP (4.6 mm ID x 15 cm L) column for 10 minutes at a flow rate of approximately 0.35 mL / min 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 correct identification and analysis was performed by self-integration using parameters known in the art.

[0219] Strong Cation Exchange Chromatography (SCEX): Injections of samples dissolved in MILLI-Q™ water were run at a flow rate of 0.4 mL / min using a gradient method starting with 100% mobile phase pH gradient buffer A to 100% mobile phase pH gradient buffer B for a total run time of 40 min on a MAbPac™ SCX-10 RS analytical column, 2.1 mm ID x 15 cm L, 5 μm column, followed by washing and re-equilibration. Peaks were manually inspected to ensure correct identification and analysis was performed by self-integration using parameters known in the art.

[0220] Hydrophobic Interaction Chromatography (HIC): Injections of samples dissolved in a diluent consisting of 750 mM ammonium sulfate, 50 mM sodium dihydrogen phosphate dihydrate, pH 6.0 (1 mg / mL) were run at a flow rate of 1 mL / min on a MAbPac™ HIC-20 HPLC column, 5 μm, 4.6 mm ID×25 cm L column for a total run time of 60 minutes using a gradient method starting with 50% mobile phase A consisting of 2 M ammonium sulfate, 50 mM sodium dihydrogen phosphate dihydrate, pH 6.0 to 100% mobile phase B consisting of 50 mM sodium dihydrogen phosphate dihydrate, pH 6.0, followed by washing and re-equilibration using 50% mobile phase A. Peaks were manually inspected to ensure correct identification and analysis was performed by self-integration using parameters known in the art.

[0221] 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 measurement instrument to ensure uniformity and then circulated and agitated by the Horiba particle size analyzer. Particle size analysis was performed using a mobile phase of isopropyl alcohol and the volume average particle size distribution was calculated.

[0222] Microflow imaging: Flow 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 dissolution method described above and diluted to 1 mg / mL in 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.

[0223] Results: The stability of the dry particles and particle suspensions with flocculants was evaluated in parallel with the protein liquid drug substance (LDS). Stability was tracked at 5, 25, and 40°C, with data collected at 7, 14 days, 1, 3, 6, 9, and 12 months. Particle stability, protein stability of the LDS, and suspensions were measured at each time and temperature. Analysis of the particles and particle suspensions confirmed that protein quality remained constant, as measured by monomer profile (SEC), maintenance of charge variant profile (CEX), isoforms / presumed oxidation (HIC), and colloidal stability (turbidity and sub-visible particles). In each case, protein feed solution was measured against the particles and particle suspensions with flocculants. Particles stored as dry powders and particle suspensions at 5, 25, and 40°C exhibited a smooth, spherical morphology. No changes in particle morphology or particle size distribution were observed at these temperatures over a 3-month period. Moisture content of the particles remained constant for all storage temperatures and lengths of time. Protein aggregate analysis was measured by SEC at 5, 25, and 40° C. for up to 12 months. The effect of excipient selection (histidine, arginine.HCl, and PS80) on the stability of hIgG particles (without flocculant) for Δ(Mon+Dim)% and Δ(Dim+Agg)% was performed for standards and stored at 40° C. for 12 months to assess flocculation over time when compared to LDS samples. As shown in FIG. 3, LDS samples stored at 40° C. for 12 months showed higher protein aggregation compared to suspensions with hIgG particles and flocculant samples stored at the same temperature (40° C., 12 months).

[0224] The lower rate of flocculation for particle suspensions with flocculant indicated improved stability compared to protein LDS. The charge variant profile of the protein was measured by SCEX at 5, 25, and 40°C for up to 90 days. No discernible changes were observed for particle suspensions with flocculant and particles that showed improved stability at 40°C compared to protein LDS. HIC was used to measure the potential oxidation of the protein. The estimated overall degree of oxidation of the protein was measured by HIC at 5, 25, and 40°C for up to 90 days. No discernible changes were observed, indicating that particle and particle suspensions with flocculant showed improved stability with respect to oxidative stress at 25 and 40°C compared to protein LDS at the same temperature and time. To further explore the protein quality of the redissolved particles in aqueous media, visible and subvisible particles were analyzed after storage at various temperatures. Upon dissolution, the protein solution was essentially free of visible and subvisible particles after storage at 40°C for up to 90 days. For particle suspensions with flocculants, protein concentration, viscosity, and injection force did not change significantly over all storage times and temperatures.

[0225] Example 5 Protein stability with aggregation agents by structural assay (days 0, 7 and 30 at 40°C): Size Exclusion Chromatography (SEC): The objective was to collect SEC data to evaluate the degradation of protein monomer in the presence of flocculant through storage at 40° C. for 30 days. SEC was performed on protein liquid drug substance (LDS), particles, and suspension with flocculant. Monomer, aggregate, and fragment data for each sample was obtained. SEC data was recorded at 0, 7, and 30 days after storage at 40° C., indicating good stability of protein monomer through treatment and storage with flocculant. The 30-day sample for LDS had poorer retention of monomer compared to particle suspension with flocculant, which each had >98% retention of monomer.

[0226] Cation Exchange Chromatography (CIEX): CIEX data was collected to assess the distribution of charge variant species and their changes throughout storage in the presence of flocculant at 40° C. for 30 days. CIEX was performed on the LDS, particles, and suspensions with flocculant. Acidic, major, and basic species data for each sample was obtained and analyzed. Selected CIEX data points highlighted the acidic shift experienced by the label formulation compared to the absence of such a shift in the suspensions after 30 days of aging. The protein from the suspensions resembled the untreated material, while the LDS experienced high degradation. CIEX data was recorded as proposed and showed good stability of the charge variants throughout storage with flocculant at 0, 7, and 30 days after storage flocculant at 40° C. The 30 day sample for LDS had dramatically lower retention of charge variant distribution compared to the particle suspensions with flocculant due to the acidic shift in the variants.

[0227] Protein Aggregation, Fragmentation, and Charge Variant Changes: Aggregation and fragmentation data were collected using size exclusion chromatography (HPLC-SEC) according to Examples 1-3 unless otherwise stated. Charge variant species distribution and their changes were collected using strong cation exchange chromatography (CIEX) according to Examples 1-3 unless otherwise stated. Results showed improved stability for suspensions with flocculants compared to the starting LDS.

[0228] Example 6 Flocculant and protein concentration studies: The average sliding force was plotted against particle suspension concentrations up to 700 mg / mL (74% protein: 2% histidine: 24% arginine: 1% PS80). 20 N (Newtons) was accepted as a reference for the typical sliding force limit.

[0229] Flocculation volume by increasing flocculant and protein concentration: Protein particle suspensions using BIgG were made at concentrations ranging from 300 to 700 mg / mL using ethyl oleate as the liquid carrier with flocculant (PS80) concentrations from 0 to 10 mg / mL and stored for 24 hours. Figure 4 shows a list of flocculant and protein concentration parameters for the flocculation volume study. Table 1 shows that as the concentrations of BIgG and PS80 increase, the flocculation volume increases. [Table 2]

[0230] Injectability by increasing flocculant and protein concentration: Protein particle suspensions using BIgG were made at concentrations ranging from 500 to 700 mg / mL using ethyl oleate as the liquid carrier with flocculant (PS80) concentrations of 0 to 10 mg / mL, using 20-gauge and 22-gauge 13 mm (half-inch) needles (Japan Bio Products) attached to 1 mL syringes. Table 2 shows that as the concentrations of BIgG and PS80 increase, the flocculation volume increases without a substantial change in injection force. [Table 3]

[0231] Injectability using caprylic triglyceride: Protein particle suspensions using BIgG were made at a concentration of 620 mg / mL (74% protein: 2% histidine: 24% arginine: 1% PS80) using caprylic triglyceride as the carrier liquid with a flocculant (PS80) concentration at 0.1 mg / mL. No change in aggregate volume was observed after 24 hours. Using a 22 gauge 13 mm (half inch) needle (Japan Bio Products) attached to a 1 mL syringe, the injection force was 5.2 N at 0.1 mL / sec as shown in Figure 5.

[0232] Aggregation volume and storage: Protein particle suspensions using BIgG were made at a concentration of 556 mg / mL (74% protein: 2% histidine: 24% arginine: 1% PS80) using ethyl oleate as the carrier liquid with aggregant (PS80) concentrations at 0 mg / mL and 10 mg / mL and stored at 4°C to 25°C for one month. As shown in Figure 6, no change in aggregate volume was observed at 4°C or 25°C after one month when PS80 was added to the suspension. Furthermore, the injection force remained essentially the same at 2.4 N (maximum 14 seconds) using a 20-gauge 13 mm (half-inch) needle (Japan Bio Products) attached to a 1 mL syringe. See Figure 7.

[0233] Injection force by needle gauge: Protein particle suspensions using BIgG were made at a concentration of 640 mg / mL (74% protein: 2% histidine: 24% arginine: 1% PS80) using ethyl oleate as the liquid carrier with a flocculant (PS80) concentration of 10 mg / mL, using 20-gauge, 25-gauge, and 27-gauge 13 mm (half inch) needles (Japan Bio Products) attached to 1 mL syringes. After 24 hours, the flocculation volume remained at 100%, and there was virtually no change in injection force for all three needle sizes (3 N for 20-gauge, 6 N for 25-gauge, and 16 N for 27-gauge). See FIG. 8.

[0234] Injection force using a 27-gauge UTW needle: Protein particle suspensions using BIgG were made at a concentration of 625 mg / mL (74% protein: 2% histidine: 24% arginine: 1% PS80) using ethyl oleate as the liquid carrier with a flocculant (PS80) concentration of 10 mg / mL, using a 27-gauge UTW, 13 mm (half-inch) needle (Japan Bio Products) attached to a 1 mL syringe. After 24 hours, the flocculation volume remained at 100% and there was virtually no change in injection force (17.4 N). Figure 9 shows the injection force in relation to injection time using a 27-gauge UTW needle at a protein concentration of 625 mg / mL.

[0235] Storage of capsules: A protein particle suspension using BIgG was made at a concentration of 556 mg / mL using ethyl oleate as the liquid carrier with a flocculant (PS80) concentration of 10 mg / mL and added to two gelatin capsules (18 mm length, Medlab) without air bubbles. After one month, the flocculation volume remained at 100% and there was no observable settling of protein particles. See Figures 10A and 10B.

[0236] Example 7 Pharmacokinetics (PK): Subcutaneous administration of Ab microparticle suspensions shows higher bioavailability than aqueous Ab injections: Composition: The scope of the study involves four cohorts with the following compositions: 1) IV Ab aqueous rituximab: 30 mg / mL 2) SC Ab aqueous: 30 mg / mL rituximab (anti-CD-20 antibody), 4 mg / mL histidine, 1 mg / mL trehalose, 0.7 mg / mL sodium chloride, and 0.6 mg / mL polysorbate 80; 3) SC Ab protein microparticle suspension: 30 mg / mL rituximab (anti-CD-20 antibody), 4 mg / mL histidine, 1 mg / mL trehalose, 0.7 mg / mL sodium chloride, and 0.6 mg / mL polysorbate 80 formulated into a particle composition and then suspended in ethyl oleate (non-aqueous carrier vehicle) with aggregating agent (PS80) at a concentration of 10 mg / mL. The concentration of the suspension was 30 mg / mL, and 4) SC suspension vehicle: ethyl oleate (non-aqueous carrier vehicle).

[0237] Animals: Female wild-type albino Sprague Dawley rats (Charles River Lab) were housed in the animal facility at Tufts University Comparative Medicine Services (Tufts CMS). All described animal uses were performed in accordance with the National Research Council's "Guide for the care and use of laboratory animals" and according to detailed written protocols approved by the Institutional Animal Care and Use Committee (IACUC).

[0238] Dosing, Study Design, and Analysis: All particle composition injections were accomplished using 100 μL Hamilton glass syringes with 27-gauge needles. No protocol was implemented to resuspend the particles in the syringes. All aqueous and non-aqueous vehicle injections were accomplished using 300 μL insulin syringes with a staked-in 30-gauge needle. No protocol was implemented to resuspend the particles in the syringes. Sprague Dawley rats were used for this study. The following study designs were used: 1) IV Ab aqueous: dose=10 mg / kg; volume=100 μL; n=5, 2) SC Ab aqueous: dose=10 mg / kg; volume=100 μL; n=5, 3) SC Ab microparticle suspension: dose=10 mg / kg; volume=100 μL; n=5, 4) SC suspension vehicle: volume=100 μL; n=3. Plasma samples were collected at 0h, 3h, 6h, 9h, 1d, 2d, 3d, 5d, 6d, 9d, 12d, (14d, 16d, 19d, 21d, and 23d. These time points were below the limit of detection of the assay utilized). Blood from each rat was collected into EDTA capillary tubes (purple) from tail vein bleeding 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) by a third-party contract laboratory. Test article aqueous rituximab SC injection: AUC5.8 (h*mg / mL), Cmax34.0 (μg / mL), Tmax57.6 (hours). Test article Rituximab microparticle suspension in ethyl oleate (EO) SC injection: AUC 6.3 (h*mg / mL), Cmax 36.7 (μg / mL), Tmax 67.2 (hours). As shown in Figure 11, the Rituximab microparticle suspension shows higher bioavailability than the aqueous Rituximab injection.

[0239] Toxicity and Local Tolerability Studies and Analysis: Injection sites were evaluated for edema (swelling) and erythema (redness) pre- and up to 3 days post-injection. Sites were scored based on the severity of the response using the Draize scoring system (maximum 0-4). No abnormal findings were observed on days 0 (pre-injection), 1 (post-injection), and 7 (post-injection) in any of the 5 animals in the composition groups (100 mg / kg, 500 mg / mL).

[0240] Erythema and scabbing: no erythema - 0; very mild erythema (barely perceptible) - 1; well defined erythema - 2; moderate to severe erythema - 3; severe erythema (beef redness) to scabbing that prevents grading of the erythema - 4.

[0241] Edema formation: no edema - 0; very mild edema (barely perceptible) - 1; mild edema (edge ​​of area well defined by a well defined bump) - 2; moderate edema (approximately 1mm bump) - 3; severe edema (more than 1mm bump and extending beyond the exposed area) - 4.

[0242] Draize Scoring: The absence or presence of findings was recorded for each animal on days 0 (pre-injection), 1 (post-injection), and 7 (post-injection). All five animals in the group had edema and erythema scores of zero over the three days following injection, indicating that the composition samples were well tolerated without toxicity.

[0243] Detailed clinical observations: Animals were removed from their cages and observations were made. These included, but were not limited to, evaluation of the skin, fur, eyes, ears, nose, oral cavity, chest, abdomen, vulva, legs and feet, respiratory and circulatory effects, autonomic effects such as salivation, nervous system effects including tremors, convulsions, responsiveness to handling, and abnormal behavior. No abnormal findings were observed on days 0 (pre-injection), 1 (post-injection), and 7 (post-injection) in any of the five animals in the composition groups (100 mg / kg, 500 mg / mL), indicating that the composition samples were well tolerated without observable toxicity.

[0244] Example 8 Immunogenicity: Subcutaneous administration of Ab microparticle suspensions shows no immune response: Compositions: The scope of the study included the following compositions: 1) SC carrier liquid control: ethyl oleate; 10 μL; 2) SC mAb aqueous: 20 mg / mL rituximab biosimilar (anti-CD20 antibody), 4 mg / mL histidine, 1 mg / mL trehalose, 0.7 mg / mL sodium chloride, and 0.6 mg / mL polysorbate 80; 10 μL; 3) SC mAb formulated into particles and then suspended in ethyl oleate (non-aqueous carrier vehicle) with a flocculating agent (PS80) at a concentration of 10 mg / mL. mAb microparticle suspension: 20 mg / mL Rituximab biosimilar (anti-CD20 antibody), 4 mg / mL histidine, 1 mg / mL trehalose, 0.7 mg / mL sodium chloride, and 0.6 mg / mL polysorbate 80; 10 μL; and 4) with four cohorts having SC resolved aqueous mAb: Rituximab biosimilar (anti-CD20 antibody); 10 μL, which was subjected to heating and agitation to generate a positive control sample.

[0245] Animals: Female wild-type Balb / c mice (Charles River Labs) were housed in the animal facility at Tufts University Comparative Medicine Services (Tufts CMS). All animal procedures were formally reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Tufts CMS.

[0246] Dosing, Study Design, and Analysis: All particle composition injections were performed using 100 μL Hamilton glass syringes with 27-gauge needles. All aqueous and non-aqueous vehicle injections were performed using 300 μL insulin syringes with staked-in 30-gauge needles. No protocol was implemented to resuspend the particles in the syringes. Balb / c female mice (Charles River Lab) at 10 weeks of age were used for this study. The following study designs were used: 1) SC carrier liquid control: volume = 10 μL; n = 10, 2) SC Rituximab aqueous: dose = 10 mg / kg; volume = 10 μL; n = 10, 3) SC Rituximab microparticle suspension: dose = 10 mg / kg; volume = 10 μL; n = 10, 4) SC disintegrated Rituximab aqueous: dose = 10 mg / kg, volume = 10 μL; n = 10. Plasma samples were collected at 0 d and 35 d. Blood from each mouse was collected into EDTA capillary tubes from a facial vein bleed at all data points. Plasma obtained from the blood was diluted 2,500-fold for analysis using the Meso Scale Discovery® electrochemiluminescence (MSD-ECL) platform. A master mix was generated with equal concentrations (0.5 μg / ml) of sulfo-TAG and biotinylated rituximab diluted in assay diluent (1% MSD Blocker A in PBS with 0.02% Tween 20). An ADA standard curve was generated by serially diluting mouse anti-rituximab antibody (GenScript A01970-40) at 1000 ng / mL, 333 ng / mL, 111 ng / mL, 37 ng / mL, 12 ng / mL, 4 ng / mL, 1 ng / mL, and 0 ng / mL. First, 50 μL of the master mix was added to each well of a round-bottom 96-well polypropylene plate. 25 μL of ADA standard or 4% plasma sample was then added to each well. Plates were sealed and incubated overnight at 4° C. with moderate shaking. 150 μL of blocking solution (3% w / v MSD ​​Blocker A in PBST) was added to each well of a 96-well streptavidin plate (MSD L55SA).The plates were sealed and incubated at room temperature with moderate shaking for at least 30 min, followed by washing with 200 μL of PBST. 50 μL of solution was transferred from each well of the polypropylene plate to the streptavidin plate. The plates were then sealed and incubated at room temperature for 1 h with shaking at 400-700 rpm, followed by three washes with 200 μL of PBST. 150 μL of 2× Read buffer was added to each well. The plates were analyzed using an MSD QUICKPLEX instrument. The results of this study show that subcutaneous administration of Ab microparticle suspension does not indicate the presence of an immune response based on repeated dose anti-drug antibodies (immunogenicity) for rituximab.

[0247] Example 9 Administration of hyaluronidase allows for an increase in volume and fluid dispersion of a composition having a flocculant administered by subcutaneous syringe injection: Similar procedures as described in Examples 1-4 were used for particle and suspension production. Particle formulation: HIgG 74% protein: 2% histidine: 24% arginine: 1% PS80 provided smooth, spherical particles with 3% particle water content. FIB-SEM shows that the particles have no internal void space. Suspension formulation: carrier liquid ethyl oleate, protein content 100 mg / mL, 1% PS80. Aggregation volume remained stable for at least 1 month at 25°C. Monomer data of SVP>2μm (1.8E5), SEC98.9% measured by Aura normalized per mg protein indicate that the particles remained stable for at least 1 month at approximately 25°C.

[0248] Composition: The scope of the study involved one pork belly sample equilibrated to physiological temperature in an 8" x 18" double bag (Savenor's, Boston) with two sets of injection sites with the following composition: Group 1 injection site for PBS solution; and Group 2 injection site for SC Ab suspension administered after hyaluronidase pre-injection: i) 2000U hyaluronidase (Creative Biomart) in 8.5mg / ml NaCl, 10mM citric acid-sodium citrate, 0.9mg / ml EDTA-2Na, 0.3mg / ml CaCl2 pH 5.2, followed by ii) SC Ab suspension. The concentration of the suspension was 100mg / mL protein. No protocol was performed to resuspend the particles in the syringe.

[0249] Dosing, Study Design, and Analysis: A 5 pound portion of pork belly with skin was allowed to reach physiological temperature. The skin side was thoroughly cleaned using alcohol and then dried using sterile gauze. No protocol was performed to resuspend particles in the syringe. While the skin was being tightened, the PBS solution (Group 1) was injected subcutaneously under the skin at a 45 degree angle using a 24 mL Henke-Ject syringe and a 27G UTW needle. The suspension was dispensed until sufficient back pressure was created to prevent further dispensing of the suspension. The volume dispensed was 1.4 mL, with an average injection rate of 0.025 mL / sec. The experiment was repeated with SC Ab suspension administered subcutaneously after hyaluronidase pre-injection (Group 2), where 1 mL of the hyaluronidase mixture was pre-injected subcutaneously under the skin at a 45 degree angle using a 1 mL tuberculin syringe and a 25G UTW needle. After a period of approximately 5-10 minutes, the SC Ab suspension was injected subcutaneously at a 45 degree angle into the same injection site using a 24 mL Henke-Ject syringe and a 25G UTW needle. The suspension was dispensed until sufficient back pressure prevented any further dispensing of the suspension. The average volume was 5.3 mL and the average injection rate was 0.045 mL / sec. Study endpoints included injection volume measurements and qualitative assessment of the injection site. All injections were performed using the same injection force.

[0250] Results: The purpose of this study was to determine the maximum feasible volume that could be dosed when given subcutaneously as a single dose. The experiment demonstrated that the injection volume of SC Ab suspension administered subcutaneously (1.4 mL total volume) could be significantly increased by pre-injection with hyaluronidase (5.3 mL total volume), and these hyaluronidase injection sites showed improved rate of injection of the Ab particle composition as determined by the appearance of skin blisters formed at the site of treatment, and reduced injection force (increased injection rate), and larger volume of injection.

[0251] Example 10 Similar procedures were used for particle and suspension production as in Examples 1-4. Particle formulation: Rituximab 160 mg / mL (98%), Histidine 2.63 mg / mL (3%), pH: 6.0. Suspension formulation: Carrier liquid ethyl oleate, protein content 620 mg / mL, solid loading 843 mg / mL.

[0252] Particle generation and droplet generation (Kinematica / 20mm) studies: with a diffuse phase:continuous phase (nBA) ratio of 1 / 200, mixing of at least 16mL:3.2L, mixer speed of (10000rpm) / 60s. Solid-liquid separation process by centrifugation and subsequent drying (air 10-75RH) provided smooth and spherical particles with a particle size distribution (D10 / D50 / D90) of 9.34 / 20.32 / 40.58μm and particle water content of 3.4% (Figure 12). FIB-SEM shows that the particles have no internal void space (Figure 13).

[0253] Fill a volume of 2.56 mL using ethyl oleate as the carrier liquid. Suspension dispersion process and parameters using RSM 5000 rpm for 120 seconds and filter (60 um). Add 10 uL of stock 100 mg / mL PS80 solution in ethyl oleate (1% PS80) to the suspension and vortex to mix. Flocculation volume remained stable for at least 3 months at 25°C. Transfer 1 mL of the suspension to a 1 mL Gerresheimer 13 mm syringe and add a 25 gauge UTW needle to the syringe. Measure syringe force at 0.05 mL / sec (51.1 mm / min) or 0.1 mL / sec (189.4 mm / min) and 0.05 mL / sec (94.6 mm / min).

[0254] Syringe Force Results. Figure 14 shows that the gliding force of a 620 mg / mL rituximab suspension is 16 N when injected at 0.05 mL / sec.

[0255] Autoinjector Force Results. A syringe was filled with a 620 mg / mL suspension and placed in a test fixture to simulate a spring-actuated autoinjector. The spring was capable of expelling the suspension from the syringe with 16 N.

[0256] Example 11 Similar procedures were used for particle and suspension production as in Examples 1-4. Particle formulation: Rituximab 70 mg / mL (80%), Histidine 2.2 mg / mL (2%), Arginine.HCl 15.3 mg / mL (18%), pH: 6.0. Suspension formulation: Carrier liquid ethyl oleate, protein content 507 mg / mL, solid loading 735 mg / mL.

[0257] Particle generation and droplet generation (Kinematica / 20mm) studies: with a diffusion phase:continuous phase (nBA) ratio of 1 / 200, mixing of at least 20mL:4L, mixer speed of (8000rpm) / 60s. Solid-liquid separation process by centrifugation and subsequent drying (air 10-75RH) provided smooth and spherical particles with particle size distribution (D10 / D50 / D90) of 6.6 / 11.4 / 19.3μm and particle water content of 3% (Figure 15). FIB-SEM shows that the particles have no internal void space (Figure 16). Monomer data of SVP>2μm (3.5E5), Pro-A, oxidation of 16.00%, SCEX (acidic / neutral / basic%) 7.7 / 82.7 / 9.6, SEC 96.6% measured by Aura normalized to per mg protein indicate that the particles remained stable for at least 3 months.

[0258] Fill a volume of 3.2 mL using ethyl oleate as the carrier liquid. Suspension dispersion process and parameters using RSM 6000 rpm for 15 min and filter (60 um). Add 10 uL of stock 100 mg / mL PS80 solution in ethyl oleate (1% PS80) to the suspension and vortex to mix. Flocculation volume remained stable for at least 3 months at 25°C. Transfer 1 mL of the suspension into a 2.25 mL Neopak syringe and add a 27 gauge STW x 1 / 2 inch needle to the syringe. Measure syringe force at 0.1 mL / sec (189.4 mm / min).

[0259] Syringe Force Results. Figure 17 shows that the gliding force of a 507 mg / mL rituximab suspension is 30 N when injected at 0.1 mL / sec.

[0260] Example 12 Similar procedures as in Examples 10 and 11 were used to generate trastuzumab particles and suspensions. Particle formulation: trastuzumab 71.4 mg / mL (71%), histidine 2.2 mg / mL (2%), arginine.HCl 15.3 mg / mL (26%), PS80 0.9 mg / mL (1%), pH: 6.0. Suspension formulation: carrier liquid ethyl oleate, protein content 507 mg / mL, solid loading 735 mg / mL.

[0261] Particle generation and droplet generation (Kinematica / 20mm) studies: with a diffuse phase:continuous phase (nBA) ratio of 1 / 200, mixing of at least 18mL:3.6L, mixer speed of (4000rpm) / 60s. A solid-liquid separation process by centrifugation and subsequent drying (air 10-75RH) provided smooth, spherical particles and SEC monomer data indicates that the particles remained stable for at least 3 months.

[0262] Aggregate Volume and Storage Results: After one month, the aggregate volume remained stable at 100% with no observable settling of protein particles.

[0263] Syringe Force Results. After 1 month, results showed no change in the gliding force of the 507 mg / mL trastuzumab suspension.

[0264] Example 13 Similar procedures as in Examples 1-4 were used for particle and suspension production. Particle formulation: HIgG 74% protein: 2% histidine: 24% arginine: 1% PS80 provided smooth, spherical particles with 3% particle water content. FIB-SEM shows that the particles have no internal void space. Suspension formulation: carrier liquid ethyl oleate, protein content 500 mg / mL, 1% PS80. Aggregate volume remained stable for at least 1 month at 25°C. Monomer data of SVP>2μm (1.8E5), SEC98.9% measured by Aura normalized per mg protein indicate that the particles remained stable for at least 1 month at approximately 25°C.

[0265] Aggregate Volume and Storage Stability Results. After 1 month at 4°C, 25°C, and 40°C, aggregate volume remained stable at 100% with no observable settling of protein particles. Storage stability of 500 mg / mL HIgG suspension was followed at 4°C, 25°C, and 40°C with data collected at 1, 2, and 3 months at concentrations of PS80 in ethyl oleate at 0 mg / mL, 0.1 mg / mL, and 1.0 mg / mL. Data showed that SVP>2 μm counts decreased with increasing concentrations of PS80 formulated in ethyl oleate after storage for up to 3 months at 4°C, 25°C, and 40°C. SVP counts decreased at least 4-fold at 1.0 mg / mL PS80 in EO for 500 mg / mL HIgG suspensions stored at 25°C and 40°C for at least 3 months.

[0266] Syringe Force Results. Transfer 1 mL of the suspension into a 1 mL Schott SyriQ BioPure syringe and add a 27 gauge ½ inch needle to the syringe. Measure the syringe force at 0.1 mL / sec (189.4 mm / min). The syringe force results show that the gliding force of a 500 mg / mL HIgG suspension when injected at 0.1 mL / sec is 16 N.

[0267] Measurements for yield stress were obtained by a parallel plate rheometer (ANTON PAAR™ MCR 92). The results show that a yield stress was observed that indicates the formation of a stable flocculated volume that allows the particle suspension to flow upon addition of the flocculant.

[0268] Example 14 Particle compositions having two or more proteins: Similar procedures as in Examples 10-12 were used to generate Rituximab and Trastuzumab suspensions. Particle formulations (Examples 11 and 12): Rituximab 70 mg / mL (80%), Histidine 2.2 mg / mL (2%), Arginine.HCl 15.3 mg / mL (18%), pH: 6.0, and Trastuzumab 71.4 mg / mL (71%), Histidine 2.2 mg / mL (2%), Arginine.HCl 15.3 mg / mL (26%), PS80 0.9 mg / mL (1%), pH: 6.0. Suspension formulation: carrier liquid ethyl oleate (2% PS80) to suspend, and vortex to mix, 221.2 mg rituximab and 310.5 mg trastuzumab for a total of 531.7 mg (protein content 400 mg / mL), particle protein percentage 70%.

[0269] Aggregate volume and suspension stability results. After one month, aggregate volume remained stable at 100% with no observable settling of rituximab and trastuzumab particles. After one month, analysis of the particles and particle suspensions confirmed that there was a slight decrease in protein quality, as measured by monomer profile by SEC, when PS80 was formulated in ethyl oleate after at least one month of storage at 25°C.

[0270] Example 15 A protein particle composition having at least two carrier liquids: Similar procedures were used for particle and suspension production as in Examples 1-4. Particle formulation: HIgG 130 mg / mL (88%), histidine 5.0 mg / mL (3%), arginine.HCl 9.8 mg / mL (7%), methionine 2.0 (1%), PS80 0.1 (1%), pH: 5.5. Suspension formulation: Carrier liquid 40 / 60 ethyl oleate: sesame oil, protein content 400 mg / mL, solids loading 452 mg / mL, flocculant: 0 or 2 mg / mL.

[0271] Particle generation and droplet generation (Kinematica / 20mm) studies: with a diffusion phase:continuous phase (nBA) ratio of 1 / 200, mixing of at least 20mL:4L, mixer speed of (8000rpm) / 60s. Solid-liquid separation process by centrifugation and subsequent drying (air 10-75RH) provided smooth and spherical particles with a particle size distribution (D10 / D50 / D90) of 6.1 / 10.1 / 15.7μm and particle water content of 3%. FIB-SEM shows that the particles have no internal void space. Monomer data of SVP>2μm (3.5E5), Pro-A, oxidation of 16.00%, SCEX (acidic / neutral / basic%) 7.7 / 82.7 / 9.6, SEC 96.6% measured by Aura normalized to per mg protein indicate that the particles remained stable for at least 3 months.

[0272] Fill a volume of 3.2 mL using 40 / 60 ethyl oleate:sesame oil as carrier liquid. Suspension dispersion process and parameters using RSM 6000 rpm for 15 minutes and filter (60 um). Add 10 uL of stock 100 mg / mL PS80 solution in ethyl oleate (1% PS80) to the suspension and vortex to mix. The aggregate volume then remained stable (74.8%) for at least 1 month at 25°C compared to the suspension without the flocculant (62.3% after 1 month). Transfer 1 mL of the suspension to a 2.25 mL Gerresheimer syringe and add a 27 gauge TW x 1 / 2 inch needle to the syringe. Measure syringe force at 0.1 mL / sec (189.4 mm / min). Store at room temperature, upright until no change in aggregate volume is observed (at least 1 month storage).

[0273] Flocculation Volume and Storage Stability Results. After one month at 25° C., the flocculation volume remained stable at 74.8% compared to the suspension without flocculant (62.3% after one month) with no observable settling of protein particles. Storage stability of the 400 mg / mL HIgG suspension was followed at 25° C. with data collected at one month at concentrations of PS80 in 40 / 60 ethyl oleate:sesame oil at 0 mg / mL and 0.1 mg / mL. Data showed that SVP>2 μm counts decreased with increasing concentrations of PS80 formulated in 40 / 60 ethyl oleate:sesame oil after storage at 25° C. for at least up to one month.

[0274] Syringe Force Results. Transfer 1 mL of the suspension into a 1 mL Gerresheimer syringe and add a 27 gauge TW x 1 / 2 inch needle to the syringe. Measure the syringe force at 0.1 mL / sec (189.4 mm / min). Syringe force results show that the gliding force of a 400 mg / mL HIgG suspension with flocculant using 40 / 60 ethyl oleate:sesame oil as the carrier liquid was found to be 20.1 N when injected at 0.1 mL / sec and 27.4 N when compared to the suspension without flocculant. Viscosity was found to be 23.3 cP compared to the suspension without flocculant (31.8 cP).

[0275] Measurements for yield stress were obtained by a parallel plate rheometer (ANTON PAAR™ MCR 92). The results show that a yield stress was observed that indicates the formation of a stable flocculated volume that allows the particle suspension to flow upon addition of the flocculant.

[0276] Example 16 Protein particle compositions with MCT as carrier liquid: The same procedures as in Examples 1-4 were used for the production of particles and suspensions. Particle formulation: HIgG 130 mg / mL (88%), histidine 5.0 mg / mL (3%), arginine.HCl 9.8 mg / mL (7%), methionine 2.0 (1%), PS80 0.1 (1%), pH: 5.5. Suspension formulation: carrier liquid MCT, protein content 400 mg / mL, solid loading 452 mg / mL, flocculant: 0 or 2 mg / mL.

[0277] Particle generation and droplet generation (Kinematica / 20mm) studies: with a diffusion phase:continuous phase (nBA) ratio of 1 / 200, mixing of at least 20mL:4L, mixer speed of (8000rpm) / 60s. Solid-liquid separation process by centrifugation and subsequent drying (air 10-75RH) provided smooth and spherical particles with a particle size distribution (D10 / D50 / D90) of 6.1 / 10.1 / 15.7μm and particle water content of 3%. FIB-SEM shows that the particles have no internal void space. Monomer data of SVP>2μm (3.5E5), Pro-A, oxidation of 16.00%, SCEX (acidic / neutral / basic%) 7.7 / 82.7 / 9.6, SEC 96.6% measured by Aura normalized to per mg protein indicate that the particles remained stable for at least 3 months.

[0278] Fill a volume of 3.2 mL using MCT as carrier liquid. Suspension dispersion process and parameters using RSM 6000 rpm for 15 min and filter (60 um). Add 10 uL of stock 100 mg / mL PS80 solution in MCT (1% PS80) to the suspension and vortex to mix. The aggregate volume then remained stable (87.6%) for at least 1 month at 25°C, compared to the suspension without the flocculant (65.1% after 1 month). Transfer 1 mL of the suspension to a 2.25 mL Gerresheimer syringe and add a 27 gauge TW x 1 / 2 inch needle to the syringe. Measure syringe force at 0.1 mL / sec (189.4 mm / min). Store at room temperature, upright until no change in aggregate volume is observed (at least 1 month storage).

[0279] Flocculation Volume and Storage Stability Results. After 1 month at 25° C., the flocculation volume remained stable at 87.6% compared to the suspension without flocculant (65.1% after 1 month) with no observable settling of protein particles. Storage stability of 400 mg / mL HIgG suspension was followed at 25° C. with data collected at 1 month at concentrations of PS80 in MCT at 0 mg / mL and 0.1 mg / mL. Data showed that after storage at 25° C. for at least up to 1 month, SVP>2 μm counts decreased with increasing concentrations of PS80 formulated in MCT.

[0280] Syringe Force Results. 1 mL of the suspension is transferred to a 1 mL Gerresheimer syringe and a 27 gauge TW x 1 / 2 inch needle is added to the syringe. The syringe force is measured at 0.1 mL / sec (189.4 mm / min). The syringe force results show that the gliding force of a 400 mg / mL HIgG suspension with flocculant using 40 MCT as the carrier liquid was found to be 31.8 N when injected at 0.1 mL / sec and 38.9 N when compared to the suspension without flocculant. The viscosity was found to be 36.9 cP compared to the suspension without flocculant (45.2 cP).

[0281] Measurements for yield stress were obtained by a parallel plate rheometer (ANTON PAAR™ MCR 92). The results show that a yield stress was observed that indicates the formation of a stable flocculated volume that allows the particle suspension to flow upon addition of the flocculant.

[0282] Incorporation by Reference All publications and patents mentioned herein are herein incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0283] Equivalent While certain aspects and embodiments of the present disclosure have been discussed, the above specification is illustrative and not restrictive. Many variations of the present disclosure will become apparent to those skilled in the art upon review of this specification and the following claims. The full scope of the present disclosure should be determined by reference to the claims, together with such variations, along with their full scope of equivalents, together with this specification.

Claims

1. A pharmaceutically effective composition, A plurality of 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, A flocculant is included, wherein the concentration of the flocculant in the composition is less than about 50 mg / mL. A composition wherein the concentration of the therapeutic biological agent or a salt thereof in the composition is greater than approximately 250 mg / mL.

2. The composition according to claim 1, wherein the flocculant is ionic.

3. The composition according to claim 2, wherein the ionic flocculant is magnesium stearate, sodium dodecyl sulfate, sodium stearate, cetyltrimethylammonium bromide, lecithin, or a combination thereof.

4. The composition according to claim 1, wherein the flocculant is nonionic.

5. The composition according to claim 4, wherein the nonionic 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 oleic glyceride, poloxamer, or a combination thereof.

6. The composition according to claim 1, wherein the flocculant is polysorbate.

7. The composition according to claim 1, wherein the flocculant is polysorbate 20, polysorbate 60, or polysorbate 80.

8. The composition according to any one of claims 1 to 7, wherein the concentration of the flocculant in the composition is about 0.1 mg / mL to about 10 mg / mL.

9. The composition according to any one of claims 1 to 7, wherein the plurality of particles have a coagulation of less than about 10% of the therapeutic biological preparation.

10. The composition according to any one of claims 1 to 7, wherein the plurality of particles have a roundness of about 0.80 to about 1.

00.

11. The composition according to any one of claims 1 to 7, wherein the plurality of particles comprise more than 60% by weight of a therapeutic biological agent.

12. The composition according to any one of claims 1 to 7, wherein the plurality of particles further comprises carbohydrates, pH adjusters, salts, chelating agents, surfactants, protein stabilizers, emulsifiers, preservatives, amino acids, antioxidants, parabens, bactericides, fungicides, preservatives, or combinations thereof.

13. The composition according to any one of claims 1 to 7, wherein the therapeutic biological preparation is an antibody or a fragment thereof.

14. The composition according to claim 13, wherein the antibody is a human antibody.

15. The composition according to claim 14, wherein the human antibody is an IgG antibody.

16. The composition according to any one of claims 1 to 7, wherein the liquid carrier is non-aqueous.

17. The composition according to claim 16, wherein the non-aqueous liquid carrier is an organic solvent.

18. The aforementioned organic solvents 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, glycoflore, diglyceride, methyl tert-butyl ether, N-methylpyrrolidone, perfluorodecalin, polyethylene glycol, 2-pyrrolidone, tetrahydrofurfuryl alcohol, diglycerides, triglycerides, medium-chain triglycerides (MCT), caproic acid, caprylic acid, capric acid, lauric acid, ethyl laureate, triglycerides of fractionated vegetable fatty acids C8 and C10, and propylene glycol diglycerides of saturated vegetable fatty acids C8 and C10. 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 (ethylThe composition according to claim 17, comprising macadamiate, trimethylolpropane triisostearate, 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, or a combination thereof.

19. The composition according to any one of claims 1 to 7, wherein the concentration of the therapeutic biological agent in the composition is greater than approximately 400 mg / mL.

20. The composition according to any one of claims 1 to 7, wherein the composition has a viscosity of less than about 100 mPa·s.

21. The composition according to any one of claims 1 to 7, wherein the flocculant increases the aggregated volume of the composition compared to a suspension of a plurality of particles in a pharmaceutically acceptable liquid carrier that does not contain a flocculant.

22. A composition for treating a disease or condition, wherein the composition is A plurality of 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, A flocculant is included, wherein the concentration of the flocculant in the composition is less than about 50 mg / mL. A composition wherein the concentration of the therapeutic biological agent or a salt thereof in the composition is greater than approximately 250 mg / mL.

23. The composition according to claim 22, wherein the disease or condition is cancer, an inflammatory disease, or an immune disease.

24. The composition according to claim 22, wherein the concentration of the flocculant in the composition is about 0.1 mg / mL to about 10 mg / mL.

25. The composition according to claim 22, wherein the flocculant is polysorbate.

26. The composition according to claim 22, wherein the flocculant is polysorbate 20, polysorbate 60, or polysorbate 80.

27. The composition according to claim 22, wherein the composition further comprises at least one hyaluronane degrading agent.

28. The composition according to claim 27, wherein the hyaluronan degrading agent is hyaluronidase.

29. A kit comprising a syringe or a portable drug delivery injection device and a composition, wherein the composition is A plurality of 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, Contains a flocculant, The concentration of the flocculant in the composition is less than about 50 mg / mL, and the concentration of the therapeutic biological preparation or its salt in the composition is greater than about 250 mg / mL. kit.

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