Injectable suspension

Abstract

The present disclosure provides high concentration 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 resuspending prior to administration.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 336,743, filed on April 29, 2022. The entire teachings of the above application are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to compositions and methods that can be used to deliver (e.g., intramuscularly or subcutaneously) biopharmaceutical products for therapy. Specifically, 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 Art

[0004] Over the past few decades, biologics, especially antibodies, have driven a paradigm shift in drug discovery and development, providing help for patients who previously had little or no treatment options. For example, current monoclonal antibody (mAb) therapies typically require large doses, which are administered by intravenous (IV) infusion in high volumes and low concentrations, which may take hours to deliver, causing patient discomfort and increasing the risk of infection. Subcutaneous (SC) injections provide a more ideal alternative for delivery because they reduce the burden on hospitals and clinical facilities, require less time, and reduce the risk of complications. However, SC injections of mAb particles require low delivery volumes, which require high concentrations, which are generally difficult to obtain. The requirement for high particle concentrations at low delivery volumes can also cause particle sedimentation, which may result in excessive injection forces and require manual stirring for particle resuspension. Therefore, there is a need for a highly concentrated low-volume injectable particle composition that does not require manual premixing or resuspension before administration. Summary of the invention

[0005] Provided herein is a pharmaceutically effective composition comprising:

[0006] a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein the particles (e.g., substantially all of the particles) comprise at least one therapeutic biologic or a salt thereof; and

[0007] A flocculant, wherein the concentration of the flocculant in the composition is less than about 50 mg / mL; and

[0008] wherein the concentration of the therapeutic biologic or salt thereof in the composition is greater than about 250 mg / mL.

[0009] In one aspect, the present disclosure provides a method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a pharmaceutically effective amount of a composition comprising:

[0010] a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein the particles (e.g., substantially all of the particles) comprise at least one therapeutic biologic or a salt thereof; and

[0011] A flocculant, wherein the concentration of the flocculant in the composition is less than about 50 mg / mL; and

[0012] wherein the concentration of the therapeutic biologic or salt thereof in the composition is greater than about 250 mg / mL.

[0013] The present disclosure also provides herein a method of administering a pharmaceutically effective composition, the method comprising:

[0014] a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein the particles (e.g., substantially all of the particles) comprise at least one therapeutic biologic or a salt thereof; and

[0015] A flocculant, wherein the concentration of the flocculant in the composition is less than about 50 mg / mL; and

[0016] wherein the concentration of the therapeutic biologic or salt thereof in the composition is greater than about 250 mg / mL.

[0017] In another aspect, the present disclosure provides a pharmaceutically effective composition comprising particles suspended in a pharmaceutically acceptable liquid carrier, wherein the particles comprise:

[0018] a first plurality of particles, the first plurality of particles comprising a first therapeutic biologic or a salt thereof;

[0019] a second plurality of particles comprising a second therapeutic biologic or a salt thereof; and

[0020] a flocculant, wherein the concentration of the flocculant in the composition is less than about 50 mg / mL;

[0021] wherein the concentration of the first therapeutic biologic and the second therapeutic biologic or salt thereof in the composition is greater than about 250 mg / mL.

[0022] The present disclosure also provides a kit, which comprises: a syringe or a portable drug delivery injection device, and a composition, wherein the composition comprises:

[0023] a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein the particles (e.g., substantially all of the particles) comprise at least one therapeutic biologic or a salt thereof; and

[0024] a flocculant, wherein the concentration of the flocculant in the composition is less than about 50 mg / mL;

[0025] wherein the concentration of the therapeutic biologic or salt thereof in the composition is greater than about 250 mg / mL.

[0026] In some embodiments, the compositions of the present invention can be used to administer suspensions of high concentration therapeutic biologic particles via syringe injection, portable drug delivery injection devices, or oral administration liquid syringe capsules without the need for manual premixing or resuspension prior to administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 An image at 1000x magnification of particles used in the compositions disclosed herein is shown.

[0028] Figure 2 Shown are images of flocculation volume in the presence and absence of different flocculants (lecithin, PS80) at various concentrations.

[0029] Figure 3 Shown is a graph indicating the stability of IgG particles alone and in suspension during storage at 40°C for 12 months.

[0030] Figure 4 Parameter plots for flocculant and protein concentration are shown.

[0031] Figure 5 A syringeability graph is shown for a suspension using caprylic triglyceride as a liquid carrier.

[0032] Figure 6 A graph showing the flocculation volume over a period of time at different temperatures.

[0033] Figure 7 A graph showing the syringeability of the suspension at different temperatures over a period of time is shown, with an average force of about 2.4N.

[0034] Figure 8 A graph of the injection force through various needle sizes is shown.

[0035] Fig. 9 Shown is a graph of injection force versus injection time using a 27 gauge UTW needle.

[0036] Fig. 10A and 10B Representative images of flocculation volume during storage of capsules over a certain period of time are shown.

[0037] Fig.11 Shown are pharmacokinetic profiles of mAb microparticle suspension (SC injection) and mAb SC injection in water solution in a cohort of rats (Sprague Dawley).

[0038] Fig.12 An image at 30 μm magnification of rituximab particles used in the compositions disclosed herein is shown.

[0039] Fig.13 An image of a rituximab particle sectioned to reveal the internal cross-section is shown.

[0040] Fig.14 Shown is a graph of the sliding force of a 620 mg / mL rituximab suspension with a flocculant upon injection.

[0041] Fig.15 An image at 30 μm magnification of rituximab particles used in the compositions disclosed herein is shown.

[0042] Fig.16 An image of a rituximab particle sectioned to reveal the internal cross-section is shown.

[0043] Fig.17 Shown is a graph of the sliding force of a 507 mg / mL rituximab suspension with a flocculant upon injection. DETAILED DESCRIPTION

[0044] Treatment of biological products, particularly monoclonal antibody (mAb) therapeutic agents, has greatly improved the treatment of human diseases. However, the delivery of these biological products has brought burdens to patients. The standard of application is usually intravenous (IV) infusion at low concentrations, which may take hours to deliver, causing discomfort to patients and increasing the risk of infection in patients. Although it is preferred to deliver subcutaneously (SC) by simple injection, the restriction of SC delivery volume (1.5-2.0mL) requires antibody concentration to be greater than 100mg / mL, which is usually not feasible. The solution concentration exceeding 100mg / mL is high viscosity, which results in extremely high injection force, and usually promotes the decomposition of therapeutic antibody compositions. The utilization of particle suspension technology can deliver>500mg / mL of treatment biological products (e.g., antibodies) concentration, while maintaining the intact structure and biological activity of treatment biological products (e.g., mAbs), therefore, by converting the delivery of treatment biological products from IV to SC, it is possible to provide advantages for patients, health care providers, payers and drug developers. However, over time, therapeutic biologic particles eventually settle or sediment out of the suspension medium (e.g., a pharmaceutically acceptable liquid carrier), resulting in high injection forces; thus, manual stirring or premixing container closure by external means (e.g., vortexing or sonication) is required to resuspend the particles prior to administration of the composition.

[0045] The present disclosure generally relates to pharmaceutically effective compositions and methods, comprising: a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein the particles (e.g., substantially all of the particles) comprise at least one therapeutic biologic or a salt thereof; and a flocculant, wherein the concentration of the flocculant in the composition is less than about 50 mg / mL. In some embodiments, the concentration of the therapeutic biologic or a salt thereof in the composition is greater than about 250 mg / mL. In some embodiments, the compositions and methods described herein further comprise administering a pharmaceutically effective amount of at least one hyaluronic acid degradation agent (e.g., hyaluronidase), which can be administered simultaneously, sequentially, or intermittently with the composition. In some embodiments, a plurality of particles and a flocculant remain substantially suspended in a non-aqueous liquid carrier for at least one month. In certain embodiments, the composition is administered by syringe injection, a portable drug delivery injection device, or an oral administration liquid syringe capsule, without 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 below about 40°C. See Examples 2-3 herein for descriptions of exemplary compositions of the present disclosure comprising a plurality of particles having a flocculant suspended in a pharmaceutically acceptable liquid carrier.

[0046] It will be readily understood that the aspects and embodiments as 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 merely represents various aspects and embodiments. In addition, without departing from the scope of the present disclosure, those skilled in the art may change the compositions and methods disclosed herein. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present disclosure belongs. All disclosures and patents mentioned herein are incorporated by reference.

[0047] definition

[0048] For the purposes of this disclosure, the following definitions will apply unless expressly stated otherwise:

[0049] Unless otherwise indicated herein or clearly contradicted by context, the terms "a", "an", "the", and similar pronouns used in the context of describing the present disclosure should be interpreted as covering both the singular and the plural. Unless otherwise indicated herein or clearly contradicted by context in other ways, all compositions described herein can be carried out in any suitable manner. Unless otherwise stated, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present disclosure, without limiting the scope of the present disclosure. Any language in this specification should not be interpreted as indicating any unclaimed element as being essential to practicing the present disclosure.

[0050] The term "about" in connection with a given numerical value (eg, for temperature and time period) is intended to include the numerical value within 10% of the specified value.

[0051] As used herein, the term "administering" or "administered" means the actual physical introduction of a composition into or onto a subject, as the case may be. Any and all methods of introducing a composition into a subject are contemplated in accordance with the present disclosure; the composition is not dependent on any particular means of introduction and should not be so construed. Means of introduction are known to those skilled in the art and are also exemplified herein.

[0052] As used herein, "alkyl" or "alkane" is a fully saturated straight or branched chain non-aromatic hydrocarbon. Typically, a straight or branched chain alkyl has 1 to about 20 carbon atoms, preferably 1 to about 10 carbon atoms, unless otherwise defined. Examples of straight and branched chain alkyls include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, tert-pentyl, neopentyl, isopentyl, sec-pentyl, 3-pentyl, sec-isopentyl, active pentyl, hexyl, heptyl, octyl, ethylhexyl, and the like.1-8Straight 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. In addition, the term "alkyl" (or "lower alkyl") as used throughout the specification, examples, and claims is intended to include "unsubstituted alkyl" and "substituted alkyl", the latter referring to an alkyl moiety having a substituent replacing hydrogen on one or more carbons of the hydrocarbon backbone. If not otherwise specified, such substituents may include, for example, alkyl, halogen, hydroxyl, carbonyl (such as carboxyl and alkoxycarbonyl, formyl or acyl), thiocarbonyl (such as thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amide, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfinamido, sulfonyl, heterocyclic, aralkyl, or aromatic or heteroaromatic moieties. It will be appreciated by those skilled in the art that the substituted portion of the hydrocarbon chain itself may also be substituted if appropriate. For example, the substituents of the substituted alkyl may include substituted and unsubstituted forms of amino, azido, imino, amide, phosphoryl (including phosphonates and phosphinates), sulfonyl (including sulfates, sulfonamides, sulfamoyl and sulfonates) and silyl, as well as ethers, alkylthio, carbonyl (including ketones, aldehydes, carboxylates and esters), -CF3, -CN, etc. Exemplary substituted alkyls are described below. Cycloalkyl may be further substituted by alkyl, alkenyl, alkoxy, alkylthio, aminoalkyl, carbonyl-substituted alkyl, -CF3, -CN, etc. In some embodiments, the term "alkyl" may mean "cycloalkyl", which refers to a non-aromatic carbocyclic ring having 3 to 10 carbon ring atoms, which are combined together to form a ring. The ring may be saturated or have one or more carbon-carbon double bonds. Examples of cycloalkyl 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, etc. or combinations thereof. Aromatic hydrocarbon solvents can be benzene, toluene, etc. or combinations thereof. Alcohol or alkyl alcohol include, for example, methanol, ethanol, propanol, butanol, pentanol (pentanol), hexanol, heptanol, octanol, decanol, amyl alcohol (amylalcohol) or combinations thereof. Alkyl ethers include methyl, ethyl, propyl, butyl, etc., for example, diethyl ether, diisopropyl ether or combinations thereof. Sulfoxides include dimethyl sulfoxide (DMSO), decyl methyl sulfoxide, tetradecyl methyl sulfoxide, and the like, or a combination thereof. The term "alkyl ketone" refers to a ketone substituted with an alkyl group, for example, acetone, ethyl methyl ketone, and the like, or a combination thereof.The term "alkyl acetate" refers to an acetate substituted by an alkyl group, for example, ethyl acetate, propyl acetate (n-propyl acetate, isopropyl acetate), butyl acetate (n-butyl acetate, isobutyl acetate, sec-butyl acetate, tert-butyl acetate), pentyl acetate (n-pentyl acetate, tert-pentyl acetate, neopentyl acetate, isopentyl acetate, sec-pentyl acetate, 3-pentyl acetate, sec-isopentyl acetate, active pentyl acetate), 2-ethylhexyl acetate, etc. or a combination thereof. The term "alkyl formates" refers to formates substituted by an alkyl group, for example, methyl formate, ethyl formate, propyl formate, butyl formate, etc. or a combination thereof. The term "trialkylamine" refers to an amino group substituted by three alkyl groups, for example, triethylamine.

[0053] As used herein, "amino acid" or "residue" refers to any naturally occurring or non-natural amino acid, any amino acid derivative, or any amino acid mimetic known in the art. L-forms and D-forms of the corresponding amino acids are included, although L-forms are generally preferred. In some embodiments, the term relates to any of the 20 naturally occurring amino acids: 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 their L-forms. In some embodiments, the amino acid side chain can be the following side chain: 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, for example, the following amino acid salt: 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, etc.

[0054] As used herein, unless the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude additional additives, components, integers or steps. The terms "include" and "comprising" can be used interchangeably. As used herein, the phrases "selected from the group consisting of...", "selected from", etc. include mixtures of the specified materials. Where numerical limitations or ranges are stated herein, the endpoints are included. In addition, all values ​​and subranges within the numerical limitations or ranges are expressly included as if expressly written herein. Unless otherwise specified, reference to a singular element does not mean "one and only one", but "one or more". Unless otherwise expressly specified, terms such as "some" refer to one or more, and singular terms such as "a", "an", and "the" refer to one or more.

[0055] The term "oligopeptide" is used to refer to peptides with fewer amino acid members, rather than polypeptides or proteins. The oligopeptides described herein generally contain about two to about 40 amino acid residues. Oligopeptides include dipeptides (two amino acids), tripeptides (three amino acids), tetrapeptides (four amino acids), pentapeptides (five amino acids), hexapeptides (six amino acids), heptapeptides (seven amino acids), octapeptides (eight amino acids), nonapeptides (nine amino acids), decapeptides (ten amino acids), undecapeptides (eleven amino acids), dodecapeptides (twelve amino acids), eicosapeptides (twenty amino acids), thirty peptides (thirty amino acids), forty peptides (forty amino acids), etc. Oligopeptides can also be classified according to molecular structure: aeruginosin, cyanopeptolin, microcystin, microviridin, microginin, anabaenopeptin and cyclamide, etc. Homologous oligopeptides are oligopeptides comprising the same amino acids. In a specific embodiment, the homo-oligopeptide comprises a 10 amino acid hexamer of poly-valine, poly-alanine and poly-glycine.

[0056] The meaning of the term "peptide" is defined as a small protein with two or more amino acids, in which the carboxyl group of one amino acid is connected to the amino group of another amino acid. Therefore, at its basic level, any type of peptide synthesis includes 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 a full-length sequence that can be translated from a gene. Additionally, as used herein, a peptide can be a subsequence or a portion of a polypeptide or protein. In certain embodiments, the peptide is composed of 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 98, 99, or 100 amino acid residues. In some embodiments, the peptide is about 30 to about 100 amino acids in length.

[0057] As used herein, the term "pharmaceutically acceptable" refers to compositions suitable for use in contact with the tissues of humans and animals within the scope of sound medical judgment without producing excessive toxicity, irritation, allergic response or other problems or complications commensurate with a reasonable benefit / risk ratio. Preferably, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal government or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeias for use in animals, and more specifically for humans.

[0058] 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 it. In certain embodiments, the sequence may be natural and non-natural. The sequence of amino acids determines the overall structure and function of the protein. In some embodiments, the protein may contain 50 or more residues. In a particular embodiment, the length of the protein may contain greater than about 101 residues. The net charge of a protein can be determined by two factors: 1) the total count of acidic amino acids relative to basic amino acids; and 2) the specific solvent pH environment that exposes positive or negative residues. As used herein, "a protein with a net positive charge or a net negative charge" refers to a protein with a net positive charge or a net negative charge under a non-denaturing pH environment. Generally, those skilled in the art will recognize that all proteins can be considered "proteins with a net negative charge", regardless of their amino acid composition, depending on their pH and / or solvent environment. For example, depending on the solvent pH, different solvents may expose negative side chains or positive side chains. The protein is preferably selected from any type of enzyme or antibody or a fragment thereof that exhibits substantially the same activity as the corresponding enzyme or antibody. Proteins can serve as structural materials (e.g., keratin), as enzymes, as hormones, as transport proteins (e.g., hemoglobin), as antibodies, or as regulatory factors for gene expression. Proteins are essential 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).

[0059] 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.

[0060] It should be understood that the specific order or hierarchy of steps in the disclosed process is an illustration of an exemplary method. Based on design preferences, it is understood that the specific order or hierarchy of steps in the process can be rearranged. Some steps can be performed simultaneously. Phrases such as "embodiments" do not mean that such embodiments are applicable to all configurations of the subject technology. The disclosure related to the embodiments can be applicable to all embodiments, or one or more embodiments. Phrases such as embodiments can refer to one or more embodiments, and vice versa.

[0061] Particles

[0062] Unless otherwise defined, all technical terms, symbols and other scientific terms used herein are intended to have the meanings commonly understood by those skilled in the art to which the present disclosure belongs. In some cases, for clarity and / or for ease of reference, the terms with commonly understood meanings are defined herein, and including such definitions herein should not necessarily be interpreted as representing that the meanings commonly understood in the art are substantially different. The techniques and procedures described or quoted herein are usually well understood, and are often adopted by those skilled in the art using conventional methods. Where appropriate, unless otherwise noted, the procedures involving the use of commercially available test kits and reagents are generally carried out according to the schemes and / or parameters defined by the manufacturer. As used herein, the phrase "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 only or exclude A; only or exclude B; only or exclude C; contain or exclude a combination of A and B; contain or exclude a combination of A and C; contain or exclude a combination of B and C; or contain or exclude a combination of A, B, and C.

[0063] As used herein, the terms "particle" or "particles" or "microparticles" are interchangeable in the broadest sense and refer to a discrete body or multiple discrete bodies. The particles described herein are round and have controlled dispersivity, with a characteristic size of submicron to tens of microns, in contrast to, for example, porous monolithic "cakes" typically produced during conventional lyophilization. This morphology allows flowable powders (e.g., characterized by a low Hausner ratio) to be obtained without post-processing. In some embodiments, the term "particle" refers to an amount of one or more proteins, e.g., a therapeutic biologic or a plurality of therapeutic biologics, whose material state is substantially solid compared to a droplet.

[0064] As disclosed herein, therapeutic biologics (also referred to as biomedical products or biopharmaceuticals) are any drug products manufactured, extracted or semi-synthesized from biological sources. Therapeutic biologics 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 various natural sources, for example, humans, animals or microorganisms, and can be produced by biotechnology methods or other technologies. In some embodiments of the present disclosure, the therapeutic biologic is an antibody or a fragment thereof. In some embodiments, the antibody is a mammalian or human antibody, for example, bovine IgG, human IgG or a monoclonal antibody (mAb). In certain embodiments, the antibody is rituximab or trastuzumab. In a specific embodiment, the therapeutic biologic is a fragment of an antibody. In some embodiments, the composition described herein comprises at least two multiple particles (e.g., a first multiple particle and a second multiple particle) suspended in a pharmaceutically acceptable liquid carrier, wherein each multiple particle comprises at least one therapeutic biologic (e.g., at least one different therapeutic biologic relative to the particles in the other multiple). In some embodiments, the compositions described herein comprise at least two pluralities of particles (e.g., a first plurality of particles and a second plurality of particles) suspended in a pharmaceutically acceptable liquid carrier, wherein each plurality of particles comprises at least one antibody, e.g., a plurality of rituximab particles and a plurality of trastuzumab particles.

[0065] The terms "antibody" and "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies, polyclonal antibodies, multivalent antibodies, and multispecific antibodies, regardless of how they are produced (i.e., using immunization, recombinant, synthetic methods). Antibodies can be gamma globulins found in blood or other body fluids of vertebrates, which act in the immune system to bind to antigens to identify and / or neutralize foreign matter. Antibodies can be designated as different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, which have heavy chains named α, δ, ε, γ, and μ, respectively. Based on differences in sequence and function, the γ class is further divided into subclasses, 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 L chain from any vertebrate species can be assigned to one of two clearly distinct types, eg, kappa and lambda, based on the amino acid sequence of its constant domain.

[0066] The immunoglobulin genes identified include kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, and numerous immunoglobulin variable region genes. In certain embodiments, the light chain is classified as kappa or lambda. In certain embodiments, the heavy chain is classified as gamma, mu, alpha, delta or epsilon, which in turn limits immunoglobulin classes IgG, IgM, IgA, IgD and IgE, respectively. In a particular embodiment of the present disclosure, the antibody is an IgG antibody.

[0067] 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 to 110 or more amino acids primarily responsible for antigen recognition. The terms "variable light" chain, domain, region, and component are used interchangeably and are abbreviated as "VL" or "V L Similarly, the terms "variable heavy" chain, domain, region and component may be used interchangeably and abbreviated as "VH" or "V H ", and refers to the heavy chain of an antibody or antibody fragment. Antibodies are typically heterotetrameric glycoproteins composed of two identical light (L) chains and two identical heavy (H) chains. Each L chain is linked to an H chain by one covalent disulfide bond. Depending on the H chain isotype, the two H chains are linked to each other by one or more disulfide bonds. Each H chain and L chain also has regularly spaced intrachain disulfide bridges. The H and L chains define a specific Ig domain. Specifically, each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) of each of the α and γ chains, and p is the same. The four CH domains of the isotype and S isotype. 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 includes an Fc portion, which includes the carboxyl terminal portion of the two H chains held together by a disulfide bond. The effector functions of antibodies (such as ADCC) are determined by sequences in the Fc region, which is also the part recognized by Fc receptors (FcR) found on certain types of cells.

[0068] As disclosed herein, the pairing of VH and VL together forms a "variable region" or "variable domain", including the amino terminal domain of the heavy chain or light chain of the antibody. The variable domain of the heavy chain can be referred to as "VH". The variable domain of the light chain can be referred to as "VL". The V domain contains an "antigen binding site", which affects antigen binding and defines the specificity of a particular antibody for its specific antigen. The V region spans about 110 amino acid residues and consists of a relatively invariant stretch of 15-30 amino acids, called the framework region (FR) (usually about 4), separated by a shorter region of extreme variability called a "hypervariable region" (usually about 3), which is usually 9-12 amino acids in length. FR largely adopts a p-sheet structure, and the hypervariable region forms a loop connecting the p-sheet structure, and in some cases forms a part of the p-sheet structure. In certain embodiments, a "hypervariable region" refers to a region in which the sequence of an antibody variable domain is highly variable and / or forms a loop defined by the structure. Typically, antibodies comprise six hypervariable regions; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0069] The terms "full-length antibody", "intact antibody" and "complete antibody" are used interchangeably herein and refer to antibodies in their substantially complete form, rather than antibody fragments as defined above. These terms particularly refer to antibodies having heavy chains that contain an Fc region. Full-length antibodies may be native sequence antibodies or antibody variants. In certain embodiments, a "complete" or "complete" antibody refers to an antibody that comprises an antigen binding site as well as CL and at least heavy chain constant domains CH1, CH2 and CH3. The constant domains may be native sequence constant domains, e.g., human native sequence constant domains or amino acid sequence variants thereof.

[0070] As indicated above, unless otherwise indicated or clearly contradicted by the context, the term antibody as used herein includes fragments of antibodies that retain the ability to specifically interact (e.g., bind) with an antigen. It has been demonstrated 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 fragment, a monovalent fragment 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) domain, or a monovalent antibody as described in WO 2007 / 059782; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting essentially of the VH and CH1 domains; (iv) a Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al., Nature 341, 544-546 (1989)), which consists essentially of a VH domain and is also known as a domain antibody (Holt et al., Trends in Biotechnology 341, 544-546 (1989)). Biotechnol. 2003 Nov; 21(11): 484-90; (vi) camelids or nanobodies (Revets et al.; Expert Opin Biol. Ther. January 2005; 5(1): 111-24 and (vii) separated complementary determining regions (CDRs). In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, the two domains can be connected using recombinant methods by a synthetic linker that enables the two domains to become a single protein chain, in which the VL region and the VH region are paired to form a monovalent molecule (referred to as a single-chain antibody or single-chain Fv (scFv)); see, for example, Revets et al.; Expert Opinion in Biotherapy. January 2005; 5(1): 111-24 and Bird et al., Science 242, 423-426 (1988). Unless otherwise specified or clearly indicated in the context, such single-chain antibodies are encompassed within the term antibody. Although such fragments are generally included within the meaning of antibodies, they are collectively and independently unique features of the present invention, exhibiting different biological properties and utilities. These and other useful antibody fragments in the context of the present invention will be further discussed herein.

[0071] As disclosed herein, "complete antibody fragments including variable domains" include Fab, Fab', F(ab')2 and Fv fragments; bifunctional antibodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed by antibody fragments. "Fab fragments" consist of the entire L chain together with the variable region domain (VH) of the H chain and the first constant domain (CH1) of one heavy chain. Each Fab fragment is monovalent with respect to antigen binding, that is, it has a single antigen binding site. "Fab' fragments" differ from Fab fragments in that they have an additional small number of residues at the carboxyl terminus of the CH1 domain including one or more cysteines from the hinge region of the antibody. Fab'-SH is the name for Fab' herein, in which the cysteine ​​residues of the constant domains carry free thiol groups. "F(ab')2 fragments" roughly correspond to two disulfide-linked Fab fragments with divalent antigen binding activity, and are still able to cross-link antigens. "Fv" is the smallest antibody fragment containing complete antigen recognition and antigen binding sites. This fragment consists of a dimer of a heavy chain variable region domain and a light chain variable region domain that are tightly non-covalently associated. In single-chain Fv (scFv) species, a heavy chain variable domain and a light chain variable domain can be covalently linked by a flexible peptide linker, so that the light chain and the heavy chain can associate into a "dimer" structure similar to the dimer structure in the double-chain Fv species. Six hypervariable loops (3 loops each for the H chain and the L chain) are derived from the folding of these two domains, which provide amino acid residues for antigen binding and give the antibody antigen binding specificity. "Single-chain Fv" is also abbreviated as "sFv" or "scFv", which is an antibody fragment comprising a VH antibody domain and a VL antibody domain connected to form a single polypeptide chain. In a specific embodiment, the scFv polypeptide further comprises a polypeptide linker between the VH domain and the VL domain that enables the scFv to form a desired structure for antigen binding. In some embodiments, a "single variable domain" is half of an Fv (comprising only three CDRs specific for an antigen) that has the ability to recognize and bind to antigen, although at a lower affinity than the entire binding site.

[0072] In some embodiments, "bifunctional antibodies" refer to antibody fragments with two antigen-binding sites, the fragments comprising 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 short linkers (about 5-10 residues) between the VH domain and the VL domain, thereby achieving inter-chain rather than intra-chain pairing of the V domain, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. In some embodiments, bifunctional antibodies can be bivalent or bispecific. In certain embodiments, bispecific diabodies are heterodimers of two "cross" sFv fragments, in which the VH domains and VL domains of the two antibodies are present on different polypeptide chains. Trifunctional antibodies and tetrafunctional antibodies are also generally known in the art.

[0073] As described herein, an "antigen-binding fragment" of an antibody comprises only a portion of an intact antibody, typically including the antigen-binding site of the intact antibody and thus retaining 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 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) a Fd fragment having VH and CH1 domains; (iv) a Fd' fragment having 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 dAb fragment consisting of VH domain; (vii) isolated CDR region; (viii) F(ab')2 fragment, a bivalent fragment comprising two Fab' fragments connected by a disulfide bridge at the hinge region; (ix) single-chain antibody molecule, e.g., single-chain Fv; scFv; (x) "bifunctional antibodies" 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), which together with complementary light chain polypeptides form a pair of antigen binding regions. In some embodiments, an "antigen binding site" generally refers to a molecule that includes at least the hypervariable region and framework region required to confer antigen binding function to the V domain. In the methods described herein, the antigen binding site can be in the form of an antibody or antibody fragment (e.g., dAb, Fab, Fd, Fv, F(ab')2 or scFv). In some embodiments, the antigen binding fragment competes with the intact antibody for antigen binding, e.g., with the intact antibody from which the fragment is derived.

[0074] The term "fragment" refers to a part or a portion of an antibody or antibody chain, wherein the amino acid residues contained in the part are less than those contained in a complete or entire antibody or antibody chain. As used herein, the term "fragment" of an antibody molecule includes an Fc fragment and an antigen-binding fragment of an antibody, for example, an antibody light chain variable domain (VL), an antibody heavy chain variable domain (VH), a single-chain antibody (scFv), a F(ab')2 fragment, a Fab fragment, a Fd fragment, a Fv fragment, a single domain antibody fragment (DAb), a one-armed (monovalent) antibody, or any antigen-binding molecule formed by a combination, assembly or conjugation of such antigen-binding fragments.

[0075] In some embodiments, the term "single-chain Fv" or "scFv" or "single-chain" antibody may refer to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Typically, the Fv polypeptide further comprises a polypeptide linker between the VH domain and the VL domain that enables the sFv to form a desired structure for antigen binding. For a review of sFv, see Pluckthun, THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, Vol. 113, Rosenburg and Moore, eds. Springer-Verlag, New York, pp. 269-315 (1994).

[0076] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical, except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies (mAbs) are highly specific for a single antigenic site or determinant on an antigen. In addition to their specificity, monoclonal antibodies are also advantageous in that they can be synthesized without being contaminated by other antibodies. Monoclonal antibodies can be prepared by hybridoma methods. Monoclonal antibodies can also be separated from phage antibody libraries using molecular engineering techniques. The monoclonal antibodies of the present disclosure can be produced by recombinant DNA methods and are sometimes referred to as "recombinant antibodies" or "recombinant monoclonal antibodies", as described herein. In some embodiments, monoclonal antibodies are antibodies of a single species, in which each antibody molecule recognizes the same epitope, because all cells producing antibodies are derived from a single B lymphocyte line. The method for producing monoclonal antibodies (mAbs) generally begins along the same route as the method for preparing polyclonal antibodies. In some embodiments, rodents (such as mice and rats) are used to produce monoclonal antibodies. In certain embodiments, rabbit, sheep or frog cells are used to produce monoclonal antibodies. The use of rats is well known and may provide certain advantages. Mice (e.g., BALB / c mice) are routinely used and typically produce a high percentage of stable fusions. In some embodiments of the present disclosure, the antibody is a monoclonal antibody. In a specific embodiment of the present disclosure, the IgG antibody is monoclonal.

[0077] In some embodiments, recombinant antibody fragments can be isolated from phage antibody libraries using techniques well known in the art. See, for example, Clackson et al., 1991, Nature 352: 624-628; 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., scFv, can be used to produce recombinant full-length immunoglobulins using methods known in the art (see, for example, Persic et al., 1997, Gene 187: 9-18).

[0078] An "isolated" antibody is a polypeptide that has been identified and separated and / or recovered from a component of its preexisting environment. Contaminant components are materials that would interfere with the therapeutic use of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.

[0079] As used herein, "human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a person. Human antibodies can be produced using various techniques known in the art, including phage display libraries. Human antibodies can be prepared by applying an antigen to a transgenic animal, which has been modified to produce such antibodies to respond to antigenic attack, but its endogenous locus has been disabled. The "humanized" form of a non-human (e.g., rodent) antibody is a chimeric antibody containing a minimal sequence derived from a non-human antibody. In most cases, a humanized antibody is a human immunoglobulin (receptor antibody), wherein the residues from the hypervariable region of the receptor are replaced by residues from the hypervariable region of a non-human species (donor antibody) such as a mouse, rat, rabbit or non-human primate with desired antibody specificity, affinity and ability. In certain embodiments, the framework region (FR) residues of a human immunoglobulin are replaced by corresponding non-human residues. In addition, a humanized antibody may be included in residues not found in a receptor antibody or a donor antibody. These modifications are carried out to further improve antibody performance. Generally, a humanized antibody will comprise substantially all of the variable domains of at least one and typically two variable domains, wherein 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 FRs of human immunoglobulin sequences. A humanized antibody will optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region.

[0080] An "affinity matured" antibody is an antibody with one or more changes in its hypervariable region(s) that result in an improvement in the antibody's affinity for the antigen compared to a parent antibody that does not have those changes. In some embodiments, an affinity matured antibody may have a micromolar affinity for the target antigen. In some embodiments, an affinity matured antibody may have a nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies are produced by procedures known in the art.

[0081] "Blocking" antibodies or "antagonist" antibodies are antibodies that inhibit or reduce the biological activity of the antigen to which they are bound. In some embodiments, blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen. As used herein, "agonist antibodies" are antibodies that simulate at least one functional activity in the functional activity of the polypeptide of interest. As used herein, the terms "inhibition", "inhibit", "inhibiting", etc., regarding protein-inhibitor (e.g., antagonist) interactions, mean that, relative to the activity or function of the protein in the absence of an inhibitor, the activity or function of the protein is negatively affected (e.g., reduced). In some embodiments, inhibition refers to the reduction of the symptoms of a disease or disease. In some embodiments, inhibition refers to the reduction of the activity of a signal transduction pathway or a signal transduction pathway. Therefore, inhibition includes, at least in part, partially or completely blocking stimulation, reducing, preventing or delaying activation, desensitization, or downregulating the amount of signal transduction or enzyme activity or protein.

[0082] "Binding affinity" 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 that reflects 1:1 interactions between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X to 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 will generally bind to the antigen slowly and tend to dissociate easily, while high-affinity antibodies will generally bind to the antigen faster and tend to maintain longer binding. A variety of methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present disclosure. "Epitope" generally refers to the portion of the antigen that binds to the antigen binding site of an antibody. In some embodiments, an epitope can be "linear" because the hypervariable loops of the antibody CDRs that form the antigen binding site bind to the sequence of amino acids in the primary protein structure. In some embodiments, the epitope is a "conformational epitope," ie, an epitope to which the hypervariable loops of a CDR bind residues as they exist in the tertiary or quaternary protein structure.

[0083] In some embodiments of the aforementioned compositions, the protein, e.g., a therapeutic biologic, is an antibody. In some embodiments, antibodies include, but are not limited to, 3F8, Abagovomab, Abatacept, Abciximab, Abituzumab, Abrezekimab, Abrilumab, Acritumomab, Actoxumab, Abituzumab, Adalimumab-adbm, Adalimumab-atto, Adalimumab-bwwb, Adecatumumab, Ado-trastuzumab, and more. emtansine, Aducanumab, Afasevikumab, Afelimomab, Aflibercept, Afutuzumab, Alacizumab pegol, ALD518, Alefacept, Alemtuzumab, Alirocumab, Altumomab pentetate, Amatuximab, Anatumomab mafenatox, andecaliximab, Anetumab ravtansine), Anifrolumab, Anrukinzumab, Ansuvimab, Apolizumab, Aprutumab ixadotin, Arcitumomab, Ascrinvacumab, Aselizumab, Atezolizumab, Atidortoxumab, Atinumab, Atlizumab, Atoltivimab, Atorolimucil, Avelumab, Azintuxizumabvedotin), Bapineuzumab, Basiliximab, Bavituximab, BCD-100, Bectumomab, Begelomab, belantamab mafodotin), Belatacept, Belimumab, Bemarituzumab, Benralizumab, Bermekimab, Bersanlimab, Bertilimumab, Besilesomab, Bevacizumab, Bevacizumab-awwb, Bezlotoxumab, Biciromab, Bimagrumab, Bimekizumab, Birtamimab, Bivatuzumab mertansine), Bleselumab, Blinatumomab, Blontuvetmab, Blosozumab, Bococizumab, Brazikumab, Brentuximab vedotin, Briakinumab, Brodalumab, Brolucizumab, Brontictuzumab, Burosumab, Cabiralizumab, Camidanlumab tesirine, Camrelizumab, Canakinumab, Cantuzumab mertansine, Cantuzumab ravtansine), Caplacizumab, Capromabpendetide), Carlumab, Carotuximab, Catumaxomab, cBR96-doxorubicin immunoconjugate, Cedelizumab, Cemiplimab, Cergutuzumab amunaleukin, Cergutuzumab amunaleukin, Certolizumab pegol, Cetrelimab, Cetuximab, Cibisatamab, Cirmtuzumab, Citatuzumab bogatox, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumab tetraxetan, Codrituzumab, Cofetuzumab pelidotin, Coltuximab ravtansine), Conatumumab, Concizumab, Cosfroviximab, Crenezumab, CR6261, Crizanlizumab, Crotedumab, Cusatuzumab, Dacetuzumab, Daclizumab, Dalotuzumab, Dapirolizumab pegol, Daratumumab, Dectrekumab, Demcizumab, Denileukin diftitox, Denintuzumab mafodotin), Denosumab, Depatuxizumabmafodotin, Derlotuximabbiotin), Detumomab, Dezamizumab, Dinutuximab, Diridavumab, Domagrozumab, Dorlimomab aritox, Dostarlimab, Drozitumab, DS-8201, Duligotumab, Dupilumab, Durvalumab, Dusigitumab, Duvortuxizumab, Ecromeximab, Eculizumab, Edobacomab, Edre colomab), Efalizumab, Efungumab, Eldelumab, Elezanumab, Elgemtumab, Elotuzumab, Elsilimomab, Emactuzumab, Emibetuzumab, Emicizumab, Enapotamab vedotin), Enavatuzumab, Enfortumab vedotin, Enlimomab pegol, Enoblituzumab, Enokizumab, Enoticumab, Ensituximab, Epitumomabcituxetan), Epoetin-alfa, Epoetin-alfa-epbx, Epratuzumab, Eptinezumab, Erenumab, Erlizumab, Ertumaxomab, Etanercept, Etanercept-szzs, Etaracizumab, Etigilimab, Etrolizumab, Evinacumab, Evolocumab, Exbivirumab, Factor VIII Fc fusion protein, Factor IX Fc fusion protein, Fanolesomab, Faralimomab, Faricimab, Farletuzumab, Fasinumab, Felvizumab, Fezakinumab, Fibatuzumab, Ficlatuzumab, Figitumumab, Filgrastim, Filgrastim-sndz, Firivumab, Flanvotumab, Fletikumab, Flotetuzumab, Flotetuzumab Fontolizumab, Foralumab, Foravirumab, Fremanezumab, Fresolimumab, Frovocimab, Frunevetmab, Fulranumab, Futuximab, Galcanezumab, Galiximab, Ganitumab, Gantenerumab, Gatipotuzumab, Gavilimomab, Gedivumab, Gemtuzumabozogamicin), Gevokizumab, Gilvetmab, Gimsilumab, Girentuximab, Glembatumumab vedotin, Golimumab, Gomiliximab, Gosuranemab, Guselkumab, Ibalizumab, IBI308, Ibritumomabtiuxetan, Icrucumab, Idarucizumab, Ifabotuzumab, Igovomab, Iladatuzumab vedotin), IMAB362, Imalumab, Imaprelimab, Imciromab, Imgatuzumab, Inclacumab, Indatuximabravtansine, Indusatumab vedotin, Inebilizumab, Infliximab, Infliximab-abda, Infliximab-dyyb, Infliximab-qbtx, Intetumumab, Inolimomab, Inotuzumabozogamicin), Ipilimumab, Iomab-B, Iratumumab, Isatuximab, Iscalimab, Istiratumab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lacnotuzumab, Ladiratuzumabvedotin, Lambrolizumab, Lampalizumab, Lanadelumab , Landogrozumab, Laprituximabemtansine, Larcaviximab, Lebrikizumab, Lemalesomab, Lendalizumab, Lenvervimab, Lenzilumab, Lerdelimumab, Leronlimab, Lesofavumab, Letolizumab, Lexatumumab, Libivirumab, Lifastuzumab vedotin), Ligelizumab, Loncastuximab tesirine, Losatuxizumab vedotin, Lilotomab satetraxetan, Lintuzumab, Lirilumab, Lodelcizumab, Lokivetmab, Lorvotuzumab mertansine, Lucatumumab, Lulizumab pegol, Lumiliximab, Lumretuzumab, Atin-Lupartumabamadotin), Lutikizumab, Maftivimab, Mapatumumab, Margetuximab, Marstacimab, Maslimomab, Mavrilimumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mirikizumab, Mirvetuximab soravtansine), Mitumomab, modotuximab, Mogamulizumab, Monalizumab, Morolimumab, Mosunetuzumab, Motavizumab, Moxetumomab pasudotox, Muromonab-CD3, Nacolomab tafenatox, Namilumab, Naptumomab estafenatox, Naratuximabemtansine), Narnatumab, Natalizumab, Navicixizumab, Navivumab, Naxitamab, Nebacumab, Necitumumab, Nemolizumab, NEOD001, Nerelimomab, Nesvacumab, Netakimab, Nimotuzumab, Nirsevimab, Nivolumab, Nofetumomabmerpentan, Obiltoxaximab, Obin utuzumab), Ocaratuzumab, ocrelizumab, Odesivimab, Odesivimab-ebgn, odulimomab, Ofatumumab, Olaratumab, Oleclumab, Olendalizumab, Olokizumab, Omalizumab, Omburtamab, OMS721, Onartuzumab, Ontuxizumab, Onvatilimab, Opicinumab, Oportuzumabmonatox), Oregovomab, Orticumab, Otelixizumab, Otilimab, Otlertuzumab, Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Panrelumab (Pamrevlumab), Panitumumab, Pankomab, Panobacumab, Parsatuzumab, Pascolizumab, Pasotuxizumab, Pateclizumab, Patritumab, PDR001, Pegfilgrastim-jmdb (Pegfilgrastim -jmdb), Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumabvedotin, Pintumomab, Placulumab, Lozalizumab zalizumab), Pogalizumab, Polatuzumab vedotin, Ponezumab, Porgaviximab, Prasinezumab, Prezalizumab, Priliximab, Pritoxaximab, Pritumumab, PRO140. Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ralpancizumab, Ramucirumab, Ranevetmab, Ranibizumab, Raxibacumab, Ravagalimab, Ravulizumab, Refanezumab, and Regaviromab avirumab), relatlimab, remtolumab, reslizumab, rilonacept, rilotumumab, rinucumab, risankizumab, rituximab, rituximab-abbs, rituximab-arrx, rituximab-pvvr, pegol-rivabazumab pegol), pegol-rivabizumab, robatumumab, Rmab, roledumab, romilkimab, romiplostim, romosozumab, rontalizumab, rosmantuzumab, rovalpituzumab tesirine, rovalpituzumab tesirine, rovelizumab, rozanolixizumab, ruplizumab, sacituzumab govitecan), Samalizumab, Samrotamab vedotin, Sapelizumab, Sarilumab, Satralizumab (SA237), Satumomab pendetinpendetide), Secukinumab, Selicrelumab, Seribantumab, Setoxaximab, Setrusumab, Sevirumab, Sibrotuzumab, SGN-CD19A, SGN-CD33A, SHP647, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab, Sirtratumab vedotin), Sirukumab, Sofituzumabvedotin, Solanezumab, Solitomab, Sonepcizumab, Sontuzumab, Spartalizumab, Stamulumab, Sulesomab, Suptavumab, Sutimlimab, Suvizumab, Suvratoxumab, Tabalumab, Tacatuzumab tetraxetan、Tadocizumab、Tafasitamab、Talacotuzumab、Talizumab、Tamtuvetmab、Tanezumab、Taplitumomab paptox、Tarextumab、Tavolimab、Tefibazumab、Telimomab aritox、Telisotuzumabvedotin), Tenatumomab, Teneliximab, Teplizumab, Tepoditamab, Teprotumumab, Tesidolumab, Tetulomab, Tezepelumab, TGN1412, Tibulizumab, Ticilimumab, Tildrakizumab, Tigatuzumab, Timigutuzumab, Timolumab, Tiragotumab, Tislelizumab, Tisotumab vedotin), TNX-650, Tocilizumab, Tomuzotuximab, Toralizumab, Tosatoxumab, Tositumomab, Tovetumab, Tralokinumab, Trastuzumab, Trastuzumab-ANNS, Trastuzumab-DKST, Trastuzumab-DTTB, Trastuzumab Enmet emtansine), Trastuzumab-PKRB, Tregalizumab, Tremelimumab, Trevogrumab, Tucotuzumabcelmoleukin, Tuvirumab, Ublituximab, Ulocuplumab, Urelumab, Urtoxazumab, Ustekinumab, Utomilumab, Vadastuximabtalirine, Vanalimab, Vandortuzumabvedotin), Vantictumab, Vanucizumab, Vapaliximab, Varisacumab, Varlilumab, Vatelizumab, Vedolizumab, Veltuzumab, Vepalimomab, Vesencumab, Visilizumab, Vobarilizumab, Volociximab, Vonlerolizumab, Vopratelimab, Vorsetuzumab mafodotin, votumumab, xentuzumab, XMAB-5574, zalutumumab, zanolimumab, zatuximab, zenocutuzumab, ziralimumab, zolbetuximab (IMAB362, claudiximab), ziv-aflibercept, or zolimomab aritox.

[0084] In some embodiments of the aforementioned compositions, the antibody is monoclonal. In certain embodiments, the monoclonal antibodies include, but are not limited to, 3F8, 8H9, abatacept, abavotumab, abciximab, abituzumab, adalimumab-adbm, adalimumab-atto, adalimumab-bwwb, alirocumab, actosumab, abituzumab, azekizumab, alirocumab, actosumab, adalimumab, adelimumab, ado-enmetuzumab, aducanumab, afacilizumab, afelimomab, aflibercept, afizumab, pegol-alasacezumab, ALD518, afacept, alemtuzumab, alirocumab, atumumab pentetate ... Anilumab, anlucrumumab (IMA-638), ansvirumab, apolizumab, acitumomab, avasinumab, aseluzumab, atezolizumab, adulumab, atenumab, atelizumab (tocilizumab), atevirumab, atumab, atomizumab, avelumab, bapinumab, basiliximab, bevacizumab, bevacizumab-awwb, BCD-100, betumumab, begolocumab, belatacept, belimumab, bemarituzumab, benralizumab, bemakinumab, bosanlimab, betilimumab, besozumab, belotosumab, bicitumumab, bimalumab, bimegizumab, botezumab, mosin-bivalizumab, brevirumab, blinatumomab monoclonal antibody, brunetumab, busecuzumab, brecuzumab, vetin-brunetumab, brianuzumab, brodalumab, bruceizumab, brunetumab, bromocriptine, cabilizumab, carrelizumab, canakinumab, mocantuzumab, recantuzumab, capacilizumab, caprocilizumab, pendetide, caprulumab, caprocilizumab, catusotuximab, cilizumab, cemiplizumab, pecilizumab, celimumab, cetuximab, cebiceptin, cetumuzumab, ch.14.18, pocitabine, citumumab, clazanizumab, cleximab, titan-clituzumab, cotrastuzumab, pertin-cofetuzumab, retin-cofetuzumab, ximab, canagliflozin, concecizumab, covecizumab, cretazumab, CR6261, lizanizumab, crotuzumab, gutuzumab, dasizumab, daclizumab, darotozumab, pegol-dapilizumab, daratumumab, detrakumab, dencezumab, denileukin, martin-denituzumab, denosumab, metuximab biotin, demostatumab, dizamizumab, denutoximab, delivuzumab, domarozumab, atoduzumab, dotalimumab, trazituzumab, duligozumab, dupilumab, durvalumab, dustuzumab, dutuximab, emtansimab, eculizumab, ebumab, edrecolomab, efalizumab,Effinguzumab, edilutuzumab, elenuzumab, egentuzumab, elotuzumab, ecitumomab, emetuzumab, imatuzumab, emtuzumab, enatumumab, vetin-ennolumab, pegolimumab, enotumumab, enokizumab, enosuzumab, entuximab, sipilimumab, epoetin alfa, epoetin alfa-epbx, epratuzumab, eprenetuzumab, erlizumab, ertusumab, etanercept, etanercept-szzs, edatuzumab, etilizumab, efretuzumab, evolocumab, ivumab, factor VIII Fc fusion protein, factor IX Fc fusion protein, fasomab, faramomab, farasimab, fatutumab, fasamumab, FBTA05, panvituzumab, fezanumab, febatuzumab, felatuzumab, fentolimumab, filgrastim, filgrastim-sndz, felivolumab, fentotumab, furelcumab, vortuzumab, fontuzumab, forlecuzumab, fulavirumab, remanezumab, fesutuzumab, floximab, fulverumab, forlanumab, vortuximab, gacanezumab, galiximab, ganituzumab, ganituzumab, ganituzumab monoclonal antibody, gatuzumab, gavitumomab, gidivolumab, gemtuzumab ozogamicin, givotumomab, gilvertuzumab, ginselumab, gemtuzumab, vetin-glentumumab, golimumab, ruximab, gosanatumab, guselkumab, ibalizumab, IBI308, ibritumomab tiuxetan, eluculumab, idarucizumab, ivabrizumab, igovomab, IMAB362, imalumab, ipilimumab, insitumomab, imatumomab, inlasumab, levofloxacin-indazumab, vetin-indutumomab, inbilizumab, infliximab, infliximab-ABDA, infliximab-DYYB, infliximab-QBTX, intumumab, inomumab, inotuzumab, ipilimumab, itumumab, isatuximab, ikalimumab, isotuzumab, isotuzumab, isotuzumab, isotuzumab, isotuzumab, ixekizumab, keliximab, labetuzumab, latuzumab, lamblizumab, lambalizumab, ranalizumab, lanalizumab, laveximab, lerezumab, lemasomumab, landalizumab, renvizumab, lenzlumab, levofloxacin, levofloxacin Mucinomab, Leonlimab, Lesovotumab, Letrozumab, Lesatumumab, Rivimab, Vitin-Rifatuzumab, Ligozumab, Lilorumab Sacliracetam, Lintuzumab, Lirilumab, Lodicuzumab, Logivimab, Mocin-Lovotuzumab, Rukazumab, Rulizumab, Ruximab, Rutuzumab, Ruticuzumab, Ruticuzumab, Matuzumab, Matuximab, Matasitumomab, Maverimumab, Matuximab, Mepolizumab, Maitalizumab, Milatuzumab, Mintumomab, Milituzumab,Socin-mituximab, Mitumomab, Motuximab, Mogalimab, Monalizumab, Moromumab, Mosunituzumab, Movituzumab, Pasuto-Mosetuxomab, Moromonab-CD3, Tanacumab, Namerucizumab, Eto-napituximab, Natuximab, Natalizumab, Nasetuximab, Naviruximab, Nacitumomab, Nebacumab, Namizumab, Nemolizumab, NEO D001, nerimumab, nevacuzumab, nevituzumab, nimotuzumab, nicerviromab, nivolumab, thionotumomab-mepetan conjugate, octosacizumab, obinutuzumab, okalutuzumab, ocrelizumab, ocivirizumab, ocivirizumab-ebgn, odumumab, ofatumumab, olatumumab, oleklumab, orendalizumab, oximab, omalizumab, obinutuzumab, OMS721, anatuzumab, entuximab, ovalimumab, opinumab, motulumab, ogavuzumab, otuzumab, otuximab, otuximab, otilizumab, oselumab, ozanezumab, paxilumab, palivizumab, panrelumab, pancomanumab, panbazumab, pasaduzumab, paxilumab, pertuximab, patelizumab, patrimonumab, PDR0 01. Pegylated filgrastim-jmdb, pembrolizumab, patemuzumab, perlacetuzumab, pertuzumab, pexelizumab, pidilizumab, vetin-pinatuzumab, pintumomab, pralumab, lozalizumab, pegalizumab, vetin-pollotuzumab, ponezumab, perveximab, pracituzumab, prilizumab, priliximab, praxilumab, PRO 140, quilizumab, lilotuzumab, latrastuzumab, ratrastuzumab, ravirizumab, rapanciclovir, ramucirumab, ranevumab, ranibizumab, raxicumab, lavagalimab, ravulizumab, refelizumab, rivavir, relalizumab, nontolumab, reslizumab, linacept, rituximab, linusuzumab, risankizumab-rzaa, rituximab, rituximab-abbs, rituximab-arrx, rituximab-pvvr, rotuximab, R-mab , roledutumab, roxithromab, romiplostim, romosomumab, rotuzumab, lomantuzumab, rovizumab, loliximab, rulizumab, gosatuzumab, samazumab, salizumab, satelizumab (SA237), satumomab pendetide, secukinumab, celiklumab, ceritutumab, setosacimab, setsutumab, sevirumab, sirolimus, SGN-CD19A, SGN-CD33A, SHP647, sifalimumab, silotuximab, sintuzumab, silizumab, sirukumab, vetin-sofotuzumab,Sulanzumab, solitotumab, sonipcizumab, sontozumab, spartalizumab, setumab, thiosomab, sutofumab, sutosumab, sutitumomab, sutosumab, taperumab, yttriumab, tadumab, tancitumomab, talaculumab, talizumab, tantovumab, tantuzumab, talaculumab, talizumab, tantovumab, tantuvumab, patamumomab, tarizumab, talixizumab, tefizumab, atemumab, tetuximab, tenectumomab, tetuximab, tenetuximab, tetuximab, tedotumomab, tertulumab, rilotumab (rilotumab) ), Tezelumab, TGN1412, Tibrizumab, Tisitumumab (Tremelimumab), Titragizumab, Tigatuzumab, Timituzumab, Temolumab, Tiragozumab, Tislelizumab, TNX-650, Tocilizumab (Atelizumab), Tocilizumab, Tolazumab, Tosatosumab, Tositumomab, Tovetuzumab, Trastuzumab, Trastuzumab-ANNS, Trastuzumab-DKST (Trastuzumab-DKST t), trastuzumab-dttb, trastuzumab enmetuzumab, trastuzumab-pkrb, TRBS07, trastuzumab, trastuzumab, trastuzumab, tumefacitin, tumefacitin, tumefacitin, tumefacitin, tumefacitin, tumefacitin, tumefacitin, tumefacitin, tumefacitin, tumefacitin, tumefacitin, tumefacitin, tumefacitin, tumefacitin, tumefacitin, tumefacitin, tumefacitin, tumefacitin, tumefacitin, tumefacitin, tumefacitin, tumefacitin, tumefacitin, tumefacitin, tumefacitin In certain embodiments, the monoclonal antibody is rituximab, rituximab-abbs, rituximab-arrx, or rituximab-pvvr, or a corresponding anti-drug antibody in a sample from a human patient. In some embodiments, the monoclonal antibody is atevimab, mateviromab, osevirumab-ebgn, or a combination thereof.

[0085] In some embodiments, the monoclonal antibody is a biosimilar. In some embodiments, the biosimilar includes but is not limited to adalimumab-adbm, adalimumab-atto, adalimumab-bwwb, bevacizumab-awwb, epoetin α-epbx, etanercept-szzs, infliximab-abda, infliximab-dyyb, infliximab-qbtx, filgrastim-sndz, ocivir monoclonal antibody-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 alpha, epoetin zeta, etanercept, filgrastim, follitropin alpha, infliximab, insulin glargine, insulin lispro, pegfilgrastim, risanzumab, 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-ANN, trastuzumab-DTTB, trastuzumab-PKRB, or trastuzumab-DKST.

[0086] 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) mutant, a multispecific antibody, a bispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, a fusion protein comprising an antigenic determinant portion of an antibody, or other modified immunoglobulin molecules comprising an antigen recognition site.

[0087] In certain embodiments, the therapeutic biologic is ledipasvir / sofosbuvir, insulin glargine, lenalidomide, pneumococcal 13-valent conjugate vaccine, fluticasone / salmeterol, elvitegravir / cobicistat / emtricitabine / tenofovir alafenamide, emtricitabine, rilpivirine and tenofovir alafenamide, emtricitabine / tenofovir alafenamide, grazoprevir / elbasvir, recombinant factor VIIa, epoetin alfa, aflibercept, or etanercept.

[0088] 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, dornasalfa, dulaglutide, ecallantide, efgartigimodalfa-fcab, elosulfase alfa alfa), Etanercept-szzs, Filgrastim, Filgrastim-sndz, Galsulfase, Glucarpidase, Idursulfase, IncobotulinumtoxinA, Interferon alpha-2b, Interferon alpha-n3, Interferon beta-1a, Interferon beta-1b, Interferon gamma-1b, Laronidase, Methoxypolyethylene glycol-epoetin beta, Metreleptin, Ocriplasmin, OnabotulinumtoxinA, Oprelvekin ), Palifermin, parathyroid hormone, Pegaspargase, Pegfilgrastim, Peginterferon alfa-2a, Peginterferon alfa-2a co-packaged with ribavirin, Peginterferon alfa-2b, Peginterferon beta-1a, Pegloticase, Rasburicase, Reteplase, Rinascept, Rimabotulinumtoxin B, Romiplostim, Sargramostim, Sebelipase alfa, Tbo-filgrastim, Tenecteplase, or Ziv-aflibercept.

[0089] The activity / unit of a protein, e.g., a therapeutic biologic in a particle as disclosed herein, is about 0.5 to about 1.0, about 0.75 to about 1.0 activity / unit, or about 0.9 to about 1.0 activity / unit. The activity is measured relative to the same protein before particle formation. In some embodiments, the activity / unit of a protein is 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 the ratio corresponds to a measure of the functional or structural aspect of the protein in the feed aqueous solution immediately before droplet formation. The numerator of the 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.

[0090] In some embodiments, substantially all of the particles have less than about 10% aggregation of the treated protein (e.g., therapeutic biologic), such as less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, substantially all of the particles have less than about 5% aggregation of the therapeutic biologic. In certain embodiments, substantially all of the particles have less than about 3% aggregation of the therapeutic biologic. In some embodiments, substantially all of the particles have less than about 1% aggregation of the therapeutic biologic. In certain embodiments, substantially all of the particles have substantially no aggregation of the therapeutic biologic. In some embodiments, substantially all of the particles have less than about 0.5% aggregation of the therapeutic biologic.

[0091] In some embodiments, substantially all of the particles have less than about 10% fragmentation of the treated protein (e.g., a therapeutic biologic). In some embodiments, substantially all of the particles have less than about 3% fragmentation of the therapeutic biologic. In certain embodiments, substantially all of the particles have less than about 1% fragmentation of the therapeutic biologic. In some embodiments, substantially all of the particles have substantially no fragmentation of the therapeutic biologic. In some embodiments, substantially all of the particles have less than about 5% fragmentation of the therapeutic biologic. Suitable methods for measuring aggregation and fragmentation of therapeutic biologics can be achieved by using size exclusion chromatography (SEC).

[0092] In certain embodiments, the composition provides a change in charge variants of less than about 5% (e.g., less than about 4%, 3%, 2%, or 1%) in a population of proteins (e.g., therapeutic biologics) compared to the therapeutic biologics prior to particle formation. In some embodiments, the particles have a change in charge variants of less than about 5% in a population of therapeutic biologics after treatment. The charge variants may be acidic, basic, or neutral, and the change may be caused by post-translational modifications at the terminal amino acid, such as asparagine deamidation or lysine glycosylation. For example, charge variants include loss of positive charge by deletion of a C-terminal lysine residue, covalent bonding of the amine moieties of two lysine amino acid residues by reducing sugars, 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) may occur by converting an asparagine residue to an aspartic acid and / or isoaspartic acid residue by a deamidation reaction.

[0093] In some embodiments, compared to an aqueous composition comprising at least one therapeutic biological product in a soluble (monomer) form before particle formation, the change in charge variants of the protein (e.g., therapeutic biological product) of substantially all particles is less than about 5%. In some embodiments, the change in charge variants of the therapeutic biological products of substantially all particles is less than about 3%. In certain embodiments, the change in charge variants of the therapeutic biological products of substantially all particles is less than about 1%. In some embodiments, substantially all particles are substantially free of any change in charge variants of therapeutic biological products. An exemplary method for measuring charge variants includes cation exchange chromatography (CIEX), wherein variants are quantitatively measured by dividing the area under the peak with the variant (e.g., acidic, alkaline or neutral population) by the cumulative area contained under all peaks in the sample spectrum. The change in the percentage of charge variant population between two samples (e.g., sample A and sample B) is calculated as the numerical difference of the corresponding percentage of population variants, i.e., by subtracting the specific variant of sample B (e.g., acidic percentage) from the specific variant of sample A (e.g., acidic percentage), or vice versa. In certain embodiments, analysis can be similarly extended to all variants within a population.

[0094] The particles according to the present disclosure are round. Circularity can be used as an indicator of the shape of the particles. The particles described herein can have a characteristic circularity, for example, having a substantially circular relative shape. This characteristic describes and defines the shape of the particles based on their circularity. When the particles have a completely circular structure, the circularity is 1.0. The 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 particles is about 0.85 to about 1.00. In some embodiments, the circularity of substantially all particles is about 0.90 to about 1.00. In certain embodiments, the circularity of substantially all particles is about 0.95 to about 1.00. In a particular embodiment, the circularity of substantially all particles is about 0.98 to about 1.00. In certain embodiments, substantially all of the particles have a circularity of about 1.00. The diameter and circularity of the particles can be determined by processing images observed under an electron microscope or a flow-type particle image analyzer. The circularity can also be determined by subjecting the particles to circularity measurements and averaging the values ​​obtained. For example, the circularity (circ) can be calculated using the following formula:

[0095]

[0096] As used herein, the term "perimeter" refers to the sum of all borders of the boundary of a closed plane figure or a two-dimensional image. As used herein, the term "area" refers to the cross-sectional area of ​​a two-dimensional image of a particle. The circularity of a particle can also be described as the ratio of the minimum size of the particle to its maximum diameter. For a perfect circle, the 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 suitable for processing images, for example, by a microscope, particularly an image obtained by a scanning electron microscope (SEM) or a transmission electron microscope (TEM). In certain embodiments, the method for measuring the circularity of a particle includes an image analysis of a scanning electron micrograph of a particle, wherein the average circularity is calculated based on the cross-sectional shape of the particle projected onto the plane of the image. Such a circularity factor can be extended to identify the corresponding circularity.

[0097] In certain embodiments of the present disclosure, the particles as described herein have a smooth, rather than ridged or wrinkled, surface morphology.The surface morphology of the disclosed particles can be readily assessed by one of ordinary skill in the art of the present disclosure using routine and standard techniques.

[0098] In certain embodiments, the particles have a diameter of about 0.1 to about 1000 μm, for example, 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 diameter of the particle is about 1 to about 100 μm, for example, about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50 to about 100 μm. In some embodiments, the diameter of the particle is about 4 to about 100 μm. In certain embodiments, the diameter of the particle is about 10 to about 100 μm. In certain embodiments, the diameter of the particle is 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 particles, wherein the average spherical radius or diameter can be calculated based on the cross-sectional area of ​​the particles projected onto the plane of the image.

[0099] As used herein, the term "dispersity index" (DI) is a parameter that characterizes the degree of unevenness of the size distribution of particles. The polydispersity index (PDI), "population dispersity" or "span" (e.g., D10, D50, D90) may also refer to a value indicating the width of the particle size distribution. The particle size distribution is reported by D10, D50, D90 and the average particle size in μm, which represent the percentage of particles less than the specified D number, such as the D10 particle size is the particle size at which 10% of the mass is composed of particles with a diameter less than this value, the D50 particle size is the particle size at which 50% of the mass is composed of particles with a diameter less than this value, and the D90 particle size is the particle size at which 90% of the mass is composed of particles with a diameter less than this value. The D10, D50 and D90 particle size distributions can be measured using a laser scattering particle size analyzer.

[0100] Particle size and dispersibility can also affect injectability, e.g., syringeability. It is generally recommended that the particles be at most 1 / 10-1 / 3 of the inner diameter of the needle. Even if a small portion of the particle population is larger than the inner diameter of the needle, it may clog the needle and cause the entire dose to be wasted. In certain embodiments of the present disclosure described herein, high concentrations of therapeutic biologics in the composition are achieved by mixing particles of various sizes.

[0101] As used herein, the terms "moisture" and "water" can be used interchangeably. In certain embodiments, the moisture content of substantially all particles is less than about 7% by weight, for example, less than about 6% by weight, 5% by weight, 4% by weight, 3% by weight, 2% by weight, 1.9% by weight, 1.8% by weight, 1.7% by weight, 1.6% by weight, 1.5% by weight, 1.4% by weight, 1.3% by weight, 1.2% by weight, 1.1% by weight, 1% by weight, 0.9% by weight, 0.8% by weight, 0.7% by weight, 0.6% by weight, 0.5% by weight, 0.4% by weight, 0.3% by weight, 0.2% by weight or 0.1% by weight. In certain embodiments, substantially all particles have a moisture content less than about 7% by weight. In certain embodiments, substantially all particles have a moisture content less than about 5% by weight. In certain embodiments, substantially all particles have a moisture content less than about 3% by weight. In certain embodiments, substantially all particles have a moisture content less than about 1% by weight. Exemplary methods for measuring moisture content include chemical titration methods, such as Karl Fischer titration involving an oven. Various solvents, including water, can also be measured using weight loss methods involving thermal excitation. Exemplary methods include thermogravimetric analysis using infrared spectroscopy (TGA-IR) or gas chromatography flame ionization detector / mass spectrometry (GC-FID / MS).

[0102] As used herein, the term "internal void space" or "internal void" is opposite to pores or porosity, is not connected to the surface of a solid (e.g., a particle), and does not affect porosity or surface area. In some embodiments, substantially all particles have less than about 10% internal void space, for example, less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% internal void space. In some embodiments, substantially all particles are substantially free of any internal void space. A suitable method for determining internal void space can be achieved by using a focused ion beam scanning electron microscope (FIB-SEM), which can be used to visualize "accessible" holes and "inaccessible" void spaces. Another method for determining internal void space can be achieved by gas displacement pycnometry, which is a common analytical technique for measuring volume using a gas displacement method. An inert gas (such as helium or nitrogen) is used as a displacement medium. The true volume is the total volume minus the volume into which the gas can enter. Density is calculated by dividing the sample weight by the true volume. The sample is sealed in an instrument chamber of known volume, an appropriate inert gas is introduced, 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 give the gas displacement density. A cross section of a typical particle of the present disclosure indicates that there is no internal void space, as shown by FIB-SEM or gas pycnometry performed using helium at a temperature of about 22°C, to provide a typical average of about 1.3 g / cm 3 The density of the sample has a standard deviation of about 0.0005 g / 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 ​​the void space, and A p is the total area of ​​the particle. The cross-section of a typical particle of the present disclosure can be obtained and characterized in a variety of ways, for example, International Application Nos. PCT / US2020 / 15957 (Publication No. WO 2020 / 160323) and PCT / US2021 / 027755 (WO 2021 / 212019), the contents of each of which are hereby incorporated by reference in their entirety.

[0103] 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 have substantially no internal void space.

[0104] In some embodiments, substantially all of the particles have greater than about 50% by weight of a protein (e.g., a 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 a therapeutic biologic). In some embodiments, substantially all of the particles have greater than about 60% by weight of a therapeutic biologic. In certain embodiments, substantially all of the particles have greater than about 70% by weight of a therapeutic biologic. In some embodiments, substantially all of the particles have greater than about 80% by weight of a therapeutic biologic. In certain embodiments, substantially all of the particles have greater than about 90% by weight of a therapeutic biologic. In certain embodiments, substantially all of the particles have greater than about 95% by weight of a therapeutic biologic. In certain embodiments, substantially all of the particles have greater than about 98% by weight of a therapeutic biologic. In some embodiments, substantially all of the particles have greater than about 99% by weight therapeutic biologic.

[0105] The term "excipient" refers to an additive in a particle formulation that can be used to achieve the desired modification of the properties or morphology of the particles. Such modifications include, but are not limited to, physical stability, chemical stability, and therapeutic efficacy. Exemplary excipients include, but are not limited to, carbohydrates, pH regulators, salts, chelating agents, surfactants, protein stabilizers, emulsifiers, antimicrobial agents, amino acids, antioxidants, parabens, bactericides, fungicides, preservatives, or combinations thereof. In some embodiments, substantially all particles further comprise one or more excipients. In some embodiments, substantially all particles further comprise carbohydrates, pH regulators, salts, chelating agents, surfactants, protein stabilizers, emulsifiers, amino acids, antioxidants, or combinations thereof. In some embodiments, substantially all particles further comprise at least one carbohydrate and amino acid. In some embodiments, substantially all particles further comprise at least one surfactant and amino acid. In certain embodiments, substantially all particles further comprise at least one amino acid.

[0106] In some embodiments, carbohydrate can be from monosaccharide, disaccharide, oligosaccharide or polysaccharide family.In some embodiments, carbohydrate is dextran, trehalose, sucrose, agarose, mannitol, lactose, sorbitol, maltose, starch, alginate, xanthan gum, galactomannan, agar, agarose or its combination.In certain embodiments, carbohydrate is dextran, trehalose, sucrose, agarose, mannitol, lactose, sorbitol, maltose or its combination.In certain embodiments, carbohydrate is trehalose, cyclodextrin, hydroxypropyl β-cyclodextrin, sulfobutyl ether β-cyclodextrin, methyl β-cyclodextrin or its combination.In a particular embodiment, carbohydrate is trehalose, sucrose, sorbitol or its combination.Based on the number of glucose monomers, three different forms of cyclodextrin can be obtained: α, β and γ.The number of glucose monomers in α, β and γ cyclodextrin is 6,7 or 8 respectively.

[0107] The terms "pH adjuster" or "buffer" or "buffer species" are used interchangeably herein in the broadest sense. In some embodiments, substantially all of the particles contain a pH adjuster. In some embodiments, substantially all of the particles contain one or more pH adjusters. In certain embodiments, the pH adjuster is acetate, citrate, glutamate, glycinate, histidine, lactate, maleate, phosphate, succinate, tartrate, bicarbonate, aluminum hydroxide, phosphoric acid, hydrochloric acid, DL-lactic acid / glycolic acid, phosphoethanolamine, tromethamine, imidazole, glycine, monosodium glutamate, sodium hydroxide, potassium hydroxide, or a combination thereof. In certain embodiments, the pH adjuster is acetate, citrate, histidine, phosphate, succinate, hydrochloric acid, sodium hydroxide, or a combination thereof. In specific embodiments, the pH adjuster is histidine, hydrochloric acid, or a combination thereof. In certain embodiments, the pH adjuster is histidine.HCl, succinate, phosphate, or a combination thereof. In some embodiments, the pH level of the composition as disclosed herein is from about 5.0 to about 8.0. In some embodiments, the pH level of the compositions as 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 particular embodiments, the pH level is about 5.0, 5.5, 6.0, 6.5, or 7.0.

[0108] 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 specific embodiments, the salt is sodium chloride, sodium hydroxide, potassium nitrate, magnesium chloride, or a combination thereof. In certain embodiments, the salt is sodium chloride.

[0109] In some embodiments, the chelating agent is edetate disodium, ethylenediaminetetraacetic acid, pentetic acid, or a combination thereof.

[0110] In some embodiments, the surfactant includes but is 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 lauryl sulfate, dioctyl sodium sulfosuccinate, sodium myristyl alcohol polyether sulfate, perfluorooctane sulfonate, alkyl ether phosphate, aerosol OT (sodium bis (2-ethylhexyl) sulfosuccinate); (iii) nonionic surfactants, such as polysorbate, polyethylene glycol tert-octylphenyl ether, lecithin, alkylphenol ethoxylates, fatty alcohol ethoxylates (octylethylene glycol monododecyl ether, cocamide diethanolamine, poloxamer (poloxamer) amer), glycerol monostearate, fatty amines, fatty acid esters of sorbitol (sorbitan monolaurate, Tween 80, Tween 20, poloxamers) (nonionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)) about:blank), for example, poloxamer 188, poloxamer 407; and (iv) zwitterionic surfactants such as cocamidopropyl hydroxysultaine and 3-[(3-cholineamidopropyl)dimethylammonio]-1-propanesulfonic acid In some embodiments, the surfactant is polysorbate, magnesium stearate, sodium lauryl sulfate, alkylphenol ethoxylate, glycerin, polyoxyethylene castor oil, docusate, sodium stearate, decyl glucoside, nonoxynol-9, cetyltrimethylammonium bromide, sodium bis(2-ethylhexyl)sulfosuccinate, lecithin, sorbitan esters, phosphatidylcholine, polyglyceryl polyricinoleate, siloxane, cetyl polyethylene glycol / polypropylene glycol-10 / 1 dimethicone triglyceride, bis-polyethylene glycol / polypropylene glycol-14 / 14 dimethicone, bis-(glyceryl / lauryl)glyceryl lauryl Simethicone (and) caprylic / capric triglyceride, hexadecyl polyethylene glycol / polypropylene glycol-10 / 1-simethicone, phospholipids or combinations thereof. In certain embodiments, the surfactant is polysorbate, docusate, poloxamer or lecithin. In certain embodiments, the surfactant is polysorbate 20, polysorbate 60 or polysorbate 80. In certain embodiments, the surfactant is polysorbate 20 or polysorbate 80, for example, Tween 20, Tween 60, Tween 80. In certain embodiments, the fatty acid ester of sorbitol is an anhydrous sorbitan ester, for example span 20, span 40, span 60 or span 80. In certain 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, a polysorbate, a poloxamer, a 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.

[0111] In some embodiments, the protein stabilizer is acetyltryptophan ester, caprylate, N-acetyltryptophan, trehalose, polyethylene glycol (PEG), poloxamer, polyvinyl pyrrolidone, polyacrylic acid, poly(vinyl) polymer, polyester, polyaldehyde, tertiary 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, polyvinyl pyrrolidone, polyacrylic acid, poly (vinyl) polymer, polyester, polyaldehyde, tertiary polymer, polyamino acid, hydroxyethyl starch, N-methyl-2-pyrrolidone, sorbitol, sucrose, mannitol, cyclodextrin, hydroxypropyl β-cyclodextrin, sulfobutyl ether β-cyclodextrin, methyl β-cyclodextrin, acetyl tryptophan ester, octanoate, N-acetyl tryptophan, propylene glycol, glucose star polymer, silicone polymer, polydimethylsiloxane, carboxymethyl cellulose, poly (glycolic acid), poly (lactic acid-co-glycolic acid), polylactic acid, polycaprolactone (PCL), polyvinyl pyrrolidone (PVP), polysucrose, dextran or a combination thereof. In specific embodiments, the protein stabilizer is trehalose, cyclodextrin, hydroxypropyl β-cyclodextrin, sulfobutyl ether β-cyclodextrin, methyl β-cyclodextrin or a combination thereof. In some embodiments, protein stabilizer is hydroxypropyl β-cyclodextrin, methyl β-cyclodextrin or a combination thereof. As described herein, the stabilizer used synonymously with the term "stabilizing agent" can be a salt, a carbohydrate, a sugar or an amino acid, preferably a carbohydrate or a sugar approved by the authorities as a suitable additive or excipient in a pharmaceutical composition. In certain embodiments, PEG is PEG 200, PEG 300, PEG 3350, PEG 8000, PEG 10000, PEG 20000 or a combination thereof. The term "stabilizer" refers to an excipient or a mixture of excipients that stabilize the physical and / or chemical properties of a protein (e.g., an antibody or antibody fragment). In some embodiments, stabilizers prevent, for example, the degradation of proteins during mixing and / or storage of particulate matter. Exemplary stabilizers include, but are not limited to, sugars, salts, hydrophobic salts, detergents, reducing agents, cyclodextrins, polyols, carboxylic acids and amino acids. As described herein, a "stable" formulation or composition refers to a particulate formulation or composition comprising particles in which the protein retains an acceptable portion of its essential physical, chemical or biological properties for an acceptable period of time.In the case of proteins, for example, exemplary methods for assessing 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 the protein is assessed by measuring the size distribution of the sample at several stages. These include, for example, storage before particle formation (assessment of feed aqueous solution), immediately after particle formation, and again after the storage period in a suspension carrier medium or in the absence of a suspension carrier medium.

[0112] 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 monoglycerides and diglycerides; lipophilic polyglycerol esters; glycerides including monoolein, diolein, monostearin, distearin, monopalmitin, and dipalmitin; glycerol-lactates of fatty acids; propylene glycol esters including propylene glycol monopalmitate, propylene glycol monostearate, and propylene glycol monooleate; sorbitan esters including sorbitan monostearate. Esters, sorbitan sesquioleate; fatty acids and soaps thereof, including stearic acid, palmitic acid and oleic acid; and mixtures thereof; monoolein, diolein, monostearin, distearin, monopalmitin and dipalmitin; glyceryl-lactate esters 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 and soaps thereof, including stearic acid, palmitic acid and oleic acid; phospholipids or a combination 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, polyoxyl 40 hydrogenated castor oil, carbomer 1342, corn oil-mono-di-triglyceride, polyoxyethylated oleylglyceride, poloxamer, or a combination 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.

[0113] In some embodiments, the antimicrobial agent is phenol, m-cresol, benzyl alcohol, 2-phenoxyethanol, chlorobutanol, neomycin, benzethonium chloride, glutaraldehyde, β-propiolactone, or a combination thereof.

[0114] In certain embodiments, 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 its various salts (arginine hydrochloride, arginine glutamate etc.) or its combination. In certain embodiments, 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 its combination. In certain embodiments, amino acid is arginine, histidine, proline, asparagine or its combination. In specific embodiments, 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.

[0115] In some embodiments, the antioxidant is glutathione, ascorbic acid, cysteine, N-acetyl-L-tryptophanate, tocopherol, histidine, methionine, pentetic acid, or a combination thereof. In some embodiments, parabens are parabens. In certain embodiments, the bactericide is benzalkonium chloride (cationic surfactant), hypochlorite, peroxide, alcohol, phenolic compound (e.g., carbolic acid), benzyl benzoate, or a combination thereof. In a particular embodiment, 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.

[0116] 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.

[0117] Multiple particles comprising at least one protein (e.g., a therapeutic biologic described herein) can be prepared in a variety of ways, and for example, International Application Nos. PCT / US2017 / 063150 (Publication No. WO 2018 / 098376), PCT / US2018 / 043774 (Publication No. WO 2019 / 023392), PCT / US2019 / 033875 (Publication No. WO 2019 / 226969), PCT / US2020 / 15957 (Publication No. WO 2020 / 160323), PCT / US2020 / 050508 (Publication No. WO 2021 / 050953), PCT / US2021 / 16878 (WO 2021 / 158959), PCT / US2021 / 018806 (WO 2021 / 168271), PCT / US2021 / 027755 (WO 2021 / 212019), and PCT / US2022 / 072755 (WO 2022 / 256840), the contents of each of which are hereby incorporated by reference in their entirety.

[0118] Although each of the elements of the present disclosure are described herein as containing multiple embodiments, it should be understood that, unless otherwise indicated, 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 different embodiment of the present disclosure.

[0119] Those of ordinary skill in the relevant art will appreciate that, in view of the information known to those of ordinary skill in the art, from the description of the disclosure contained herein, other suitable modifications and adaptations of the compositions described herein will be apparent and may be made without departing from the scope of the disclosure or any embodiment thereof.

[0120] Pharmaceutical compositions of the present disclosure

[0121] Injectable particle suspensions can show sedimentation changes, which may affect the use or application of pharmaceutical compositions. Specifically, high concentration, low volume and low syringe force injectable particle suspensions for the treatment of biological products will precipitate or settle out from the suspension medium within a certain period of time, so it is necessary to premix or resuspend before injection. The sedimentation of high particle concentration under low delivery volume may also lead to high injection force, and propagate the decomposition of the treatment biological product in the composition. As used herein, the term "sedimentation" or "sedimentation" refers to the process of precipitation or dense deposition of particles at the bottom of container closure (for example, vial, cartridge, syringe, portable drug delivery injection device, etc.). The precipitation of densely deposited particles can usually cause excessive injection force or syringe clogging, and it is necessary to manually stir or premix before applying the composition.

[0122] Flocculation or "particle flocculation" is a phenomenon caused by the interaction between interfacial chemistry and environmental conditions that control particle-particle interactions. As used herein, the flocculation volume 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). Based on the sedimentation characteristics of the particle agglomerates, the flocculation volume can decrease over time. In a flocculated suspension, particles experience collisions, resulting in particle agglomerations or agglomerates, which can increase the size, for example, the flocculation volume increases. The term "agglomerate" refers to loosely coagulated clusters of particles. The loose network of particle agglomerates maintains sufficient particle-particle distance to prevent particle sedimentation that may result in high injection forces. When the flocculation volume is equal to 1, the yield stress (the stress at which the flocculation volume will undergo deformation) can be measured to indicate the shear stress required to make the suspension flow. The yield stress measurement can be performed by a parallel plate rheometer (ANTON PAAR TM MCR 92). An increase in yield stress indicates an increase in agglomerates. Other methods for measuring agglomerates include, but are not limited to, laser diffraction, relaxation NMR, microscopy, and small angle scattering.

[0123] In the compositions disclosed herein, the process of agglomeration can be controlled by using a flocculant. A flocculant or flocculating agent is a chemical substance that promotes flocculation by agglomerating suspended particles in a liquid, thereby forming agglomerates. In non-aqueous particle suspensions, the breakup and redispersion of agglomerates is determined by the shear stress applied to the flocculated particle system. Shear stress is caused by forces acting on the surface of the material, which can cause deformation. In these systems, shear can be attributed to the movement of particles or agglomerates through a velocity gradient in a non-aqueous suspension medium or near any interface (e.g., container closure, other particles, etc.). Shear stress can be introduced into the flocculation volume, for example, mechanically, and can be controlled experimentally.

[0124] The compositions disclosed herein contain flocculants and are generally high concentration, low volume and low syringe force injectable particle suspensions of therapeutic biologics that allow for administration without manual stirring or premixing prior to injection or administration of the composition. The compositions beneficially maintain a stable flocculated volume that avoids particle sedimentation that could cause clogging of a syringe, portable drug delivery injection device, or oral administration liquid injector capsule.

[0125] In certain embodiments, the present disclosure relates to a composition, e.g., a pharmaceutical composition, comprising a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein the particles (e.g., substantially all of the particles) comprise a therapeutic biologic (e.g., a therapeutic biologic disclosed herein) and a flocculant (e.g., a flocculant disclosed herein).

[0126] As used herein, the phrase "pharmaceutically acceptable liquid carrier" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid, diluent, excipient or solvent. Each carrier must be "acceptable" in the sense that it is compatible with the other ingredients of the composition and is harmless to the patient. Those skilled in the art can dilute or increase the volume of the therapeutic biological product with inert materials in the particles. These diluents can include carbohydrates, especially trehalose, mannitol, lactose, anhydrous lactose, cellulose, sucrose, modified dextran and starch. Certain inorganic salts can also be used as fillers, including tricalcium phosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

[0127] In certain embodiments, the pharmaceutical composition for administration includes a non-aqueous solution of an active therapeutic biological product in a water-soluble form. Preferably, the suspension of the active therapeutic biological product 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. In the case of injection, appropriate viscosity can be maintained by maintaining the required particle size and using a flocculant. Prevention of the effect of microorganisms can be achieved by a variety of antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.).

[0128] The present disclosure includes the use of "pharmaceutically acceptable salts" or "salts" of therapeutic biological products in the compositions of the present disclosure. As used herein, the term "pharmaceutically acceptable salts" 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 certain embodiments, pharmaceutically acceptable salt forms may include a form in which the ratio of the molecules comprising the salt is not 1:1. For example, the salt may include more than one inorganic or organic acid molecule per base molecule, such as two hydrochloric acid molecules per therapeutic biological product molecule. As another example, the salt may include less than one inorganic or organic acid molecule per base molecule, such as two therapeutic biological product molecules per tartaric acid molecule. In certain embodiments, the salts contemplated by the present disclosure include, but are not limited to, arginine, phenylethylamine, benzathine, betaine, calcium hydroxide, choline, danol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, or zinc salts. In some embodiments, the salts contemplated by the present disclosure include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts. In further embodiments, the salts contemplated by the present disclosure include, but are not limited to, alkyl, dialkyl, trialkyl, or tetraalkylammonium salts. Pharmaceutically acceptable salts may also exist as various solvates, such as with water or ethanol, etc. Mixtures of such solvates may also be prepared. In some embodiments, the protein composition may be formulated as a neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins) and are formed with inorganic acids such as hydrochloric acid or phosphoric acid or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0129] In some embodiments, the particles can be suspended in a non-aqueous liquid carrier to form a non-aqueous pharmaceutically acceptable composition. Importantly, the process of producing a non-aqueous composition with at least one therapeutic biological product does not significantly change the structure or biological activity of the biological product as 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.

[0130] In some embodiments, the organic solvent comprises 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, dimethyl isosorbide, dimethyl sulfoxide, glycogen, diethylene glycol dimethyl ether, methyl tert-butyl ether, N-methylpyrrolidone, perfluorodecalin, polyethylene glycol, diols, 2-pyrrolidone, tetrahydrofurfuryl alcohol, diglycerides, triglycerides, medium chain triglycerides (MCT), caproic acid, caprylic acid, capric acid, lauric acid, lauryl ethyl ester, fractionated vegetable fatty acid C8 and C10 triglycerides, saturated vegetable fatty acid C8 and C10 propylene glycol diesters (PGD), ethyl oleate, ethyl caprate, dibutyl adipate, fatty acid esters, caproic acid, caprylic acid, triacetin, diethylene glycol monoether, γ-butyrolactone, eugenol, cloves Bud oil, citral, limonene, polyoxyl 40 hydrogenated castor oil, polyoxyl 35 castor oil, simple alcohols such as ethanol, caprylic alcohol, hexanol, decanol, propanol, butanol, gamma-butyrolactone, tocopherol, octafluoropropane, (perfluorohexyl) octane, n-acetyl tryptophan, ethyl laurate, methyl caprylate, ethyl caprylate, methyl caprate, methyl myristic acid, methyl oleate, methyl linoleate, dimethyl adipate, dibutyl suberate, diethyl sebacate, macadamia oil The invention relates to a kind of fatty acid, which is a kind of fatty acid that is used in the manufacture of the present invention. The fatty acid that is used in the manufacture of the present invention is ethyl ester, trimethylolpropane triisostearate, isopropyl laurate, isopropyl myristate, diethyl succinate, polysorbate, ethanolamine, propionic acid, citral, anisole, anethole, benzaldehyde, linalool, caprolactone, phenol, thioglycerol, dimethylacetamide, diethylene glycol monoethyl ether, glyceryl acetone acetal, dimethyl isosorbide, ethyl formate, ethylhexyl acetate, propylene glycol dicaprylate, caprylic triglyceride, ethyl linoleate, ethyl linolenate, or a combination thereof. Medium chain triglycerides (MCT) contain fatty acids with a chain length of 6-12 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, macadamia oleate, ethyl caprate, diethyl succinate, diethylene glycol monoethyl ether, propylene glycol dicaprylate, caprylic triglyceride, ethyl linoleate, ethyl linolenate, medium chain triglycerides (MCT), sesame oil, propylene glycol diesters of saturated vegetable fatty acids C8 and C10 (PGD), triacetin, or a combination 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 a combination thereof. In some embodiments, the organic solvent is ethyl oleate and sesame oil.

[0131] In some embodiments, the concentration of the therapeutic biologic in the composition disclosed herein is greater than about 250 mg / mL, for example, greater than about 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, or 800 mg / mL. In specific 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.

[0132] In compositions as described herein, the process of agglomeration can be controlled by using a flocculant, which can allow the particle composition to be applied without manual stirring or premixing before injection or application. The flocculant can be nonionic or ionic. Typically, ionic flocculants interact with charged particles, for example, anionic flocculants interact with positively charged particles, and cationic flocculants interact with negatively charged particles. In some embodiments, the flocculant is ionic. In some embodiments, the ionic flocculant is magnesium stearate, sodium lauryl sulfate, sodium stearate, hexadecyl trimethyl ammonium bromide, lecithin or a combination thereof. In a specific embodiment, the flocculant is nonionic. As disclosed herein, nonionic flocculants interact with charged particles. In certain embodiments, the nonionic flocculant is polysorbate (polysorbate 80, polysorbate 60, polysorbate 20, for example, Tween 80, Tween 60, Tween 20), alkylphenol ethoxylate, glycerol, polyoxyethylene castor oil, docusate, decyl glucoside, nonoxynol-9, sorbitan ester, sorbitan monooleate, ethanolamine, polyoxyethylene 35 castor oil, polyoxyethylene 40 hydrogenated castor oil, carbomer 1342, corn oil-mono-di-triglyceride, polyoxyethylene oleic acid glyceride, poloxamer or its combination. In certain embodiments, the nonionic flocculant is polysorbate, alkylphenol ethoxylate, sorbitan ester, poloxamer or its combination. In certain embodiments, the nonionic flocculant is polysorbate or sorbitan ester. In certain embodiments, adding a flocculant to a particle suspension does not significantly increase the viscosity of the composition. As disclosed herein, adding a flocculant to a suspension of particles increases the flocculated volume of the suspension of particles (see, e.g., Figure 4 and Figure 6In some embodiments, the addition of a flocculant to a suspension of particles increases the stability of the flocculated volume for at least one month (see, e.g., Example 6). In some embodiments, the addition of a flocculant to a suspension of particles decreases the syringe force (see, e.g., Examples 16 and 17). In some embodiments, the addition of a flocculant to a suspension of particles improves the pharmacokinetics (PK) of microparticles (e.g., Ab microparticles) administered (e.g., subcutaneously) in suspension and exhibits a higher bioavailability than administration of an aqueous Ab solution (e.g., subcutaneously) (see, e.g., Example 16). Fig.11 In some embodiments, adding a flocculant to the suspension of particles improves the stability of the protein in the particles (see, e.g., Figure 3 ).

[0133] In some embodiments, the compositions described herein use a concentration of flocculant less than about 50 mg / mL in the composition, 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 a specific embodiment, 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.

[0134] Viscosity plays an important role in the handling and administration of injectable products. For suspension products, high viscosity drug products may be difficult to deliver through a needle (e.g., a 27-gauge needle) because more force is required to actuate the injection device, e.g., a syringe, a portable drug delivery injection device, or an oral administration liquid syringe capsule. Alternatively, using a larger gauge needle or requiring a longer injection time may reduce patient compliance with therapy.

[0135] The term "viscosity" is used to describe the property of a fluid to resist shear flow. For the purposes of this disclosure, viscosity can be determined using a rheometer (e.g., an AR-G2 rheometer (TA Instruments, USA), equipped with a cone and plate (2° / 40 mm), at a specified shear rate at 25°C). In certain embodiments, viscosity is measured at a shear rate in a Newtonian regime. In some embodiments, viscosity is measured at 100s -1 or greater shear rate (e.g., at 1000 s -1 or more than 1000s-1 or more than 10,000s -1 As used herein, the term "low viscosity" describes a composition, such as a liquid carrier, having a viscosity of less than about 100 mPa·s.

[0136] In some embodiments, the viscosity of the composition is 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 (one millipascal-second). Mixtures of liquids can also be used to control viscosity. The units "mPa·s" and "cP" are used interchangeably in the broadest sense.

[0137] In some embodiments, the viscosity of the composition is less than about 100 mPa s. In some embodiments, the viscosity of the composition is less than about 80 mPa s. In certain embodiments, the viscosity of the composition is less than about 50 mPa s. In some embodiments, the viscosity of the composition is less than about 40 mPa s. In certain embodiments, the viscosity of the composition is less than about 30 mPa s. In certain embodiments, the viscosity of the composition is less than about 25 mPa s. In some embodiments, the viscosity of the composition is less than about 20 mPa s. In certain embodiments, the viscosity of the composition is less than about 15 mPa s. In some embodiments, the viscosity of the composition is less than about 10 mPa s. In certain embodiments, the viscosity of the composition is less than about 5 mPa s.

[0138] As described herein, higher flocculation volume indicates increased agglomerates or less particle sedimentation, and therefore, due to the more uniform dispersion of particles in the liquid carrier, injection force performance is improved. In some embodiments, after initial mixing, the flocculation volume of the composition is greater than about 50%, for example, 60%, 70%, 75%, 80%, 85%, 90%, 95% or greater than about 98% after initial mixing. In some embodiments, after initial mixing, the flocculation volume of the composition is greater than about 70%. In certain embodiments, after initial mixing, the flocculation volume of the composition is greater than about 80%. In a particular embodiment, after initial mixing, the flocculation volume of the composition is greater than about 85%. In certain embodiments, after initial mixing, the flocculation volume of the composition is about 100%. Initial mixing refers to homogenizing the initial uneven particle suspension with the flocculant by entrainment motion, thereby producing a flocculation volume.

[0139] In some embodiments, after at least one month of storage in a closed container at a temperature below about 40°C, the flocculated volume is reduced by less than about 10%, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%. In some embodiments, after at least one month of storage in a closed container at a temperature below about 40°C, the flocculated volume is reduced by less than about 7%. In certain embodiments, after at least one month of storage in a closed container at a temperature below about 40°C, the flocculated volume is reduced by less than about 5%. In some embodiments, after at least one month of storage in a closed container at a temperature below about 40°C, the flocculated volume is reduced by less than about 3%. In certain embodiments, after at least one month of storage in a closed container at a temperature below about 40°C, the flocculated volume is reduced by less than about 1%. In some embodiments, the plurality of particles and the flocculant remain substantially suspended in the liquid carrier for at least one month. In some embodiments, the flocculated volume of the composition is substantially the same over at least one month.

[0140] In certain embodiments, insoluble particulate matter having a characteristic size greater than or equal to about 100 μm persists when dissolved in an aqueous liquid and is referred to as visible particles (VP). In specific embodiments of the 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 aqueous body fluid distributed in an extracellular compartment, e.g., extracellular fluid, interstitial fluid, intravascular fluid (blood, plasma, and lymph), and cerebrospinal fluid.

[0141] In some embodiments, insoluble particulate matter having a characteristic size of about 1 μm to about 100 μm persists when dissolved in an aqueous liquid, referred to as subvisible particles (SvP). SvP is present in an amount of about 0 to 100,000,000 / mL, e.g., about 0 to about 10,000,000 / mL, about 0 to about 1,000,000 / mL, about 0 to about 500,000 / mL, about 0 to about 100,000 / mL, about 0 to about 50,000 / mL, about 0 to about 10,000 / mL, about 0 to about 6,000 / mL, about 0 to about 1,000 / mL, about 0 to about 600 / mL, about 0 to about 250 / mL, about 0 to about 100 / mL, about 0 to about 60 / mL, or about 0 to about 10 / mL. In some embodiments, the count of particles with a characteristic size greater than or equal to 10 μm is about 0 to about 6,000 / mL, for example, about 0 to about 1,000 / mL, about 0 to about 100 / mL, about 0 to about 10 / mL, about 0 to about 5 / mL, about 0 to about 3 / mL, or about 0 to about 1 / mL. In certain embodiments, the count of particles with a characteristic size greater than or equal to 25 μm is about 0 to about 600 / mL, for example, about 0 to about 100 / mL, about 0 to about 10 / mL, about 0 to about 3 / mL, about 0 to about 1 / mL, about 0 to about 0.5 / mL, or about 0 to about 0.1 / mL. An exemplary method of measuring SvP includes analyzing 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, when dissolved in an aqueous liquid, the composition has a concentration of insoluble subvisible particles (SvP) of particles greater than about 10 μm of about 0 / mL to about 100,000,000 / mL. In certain embodiments, when dissolved in an aqueous liquid, the composition has a concentration of insoluble subvisible particles (SvP) of particles greater than about 10 μm of about 0 / mL to about 6000 / mL. In a specific embodiment, when dissolved in an aqueous liquid, the composition has a concentration of insoluble subvisible particles (SvP) of particles greater than about 25 μm of about 0 / mL to about 600 / mL. In certain embodiments, the composition is substantially free of insoluble subvisible particles (SvP) when dissolved in an aqueous liquid.

[0142] In certain embodiments, the composition, when dissolved in an aqueous liquid, has a concentration of insoluble subvisible particles (SvP) having a characteristic size greater than about 10 μm of about 0 / mL to about 100,000,000 / mL. In some embodiments, the composition, when dissolved in an aqueous liquid, has a concentration of insoluble subvisible particles (SvP) having a characteristic size greater than about 10 μm of about 0 / mL to about 6000 / mL. In some embodiments, the composition, when dissolved in an aqueous liquid, has a concentration of insoluble subvisible particles (SvP) having a characteristic size greater than about 25 μm of about 0 / mL to about 600 / mL. In specific embodiments, the aqueous liquid is water, an aqueous buffer, or a physiologically relevant aqueous liquid.

[0143] In some embodiments, insoluble particulate matter with a characteristic size of about 100 nm to about 1 μm persists when dissolved in an aqueous liquid, referred to as submicron particles (SMPs), and sometimes also referred to as nanoparticles. The presence of such SMPs is believed to contribute to immunogenicity and should therefore be avoided to minimize such effects. Quantitatively, SMPs are measured at about 0 to 5×10 12 / mL, for example, about 0 to about 0.5×10 12 / mL, about 0 to about 50×10 9 / mL, about 0 to about 10×10 9 / mL, about 0 to about 5×10 9 / mL, about 0 to about 0.5×10 9 / mL, about 0 to about 50×10 6 / mL, about 0 to about 1×10 6 / mL, about 0 to about 500,000 / mL, about 0 to about 200,000 / mL, about 0 to about 100,000 / mL, about 0 to about 10,000 / mL, about 0 to about 5000 / mL, or about 0 to about 1000 / mL. Exemplary methods for quantitatively measuring SMP include using NanoSight, microfluidic imaging system, asymmetric field flow fractionation coupled with multi-angle laser scattering (AF4MALS), dynamic light scattering (DLS), or FLOWCAM after reconstitution and dilution of the therapeutic biologic to a standard concentration, for example, about 100 mg / mL, about 1 mg / mL, or about 1 μg / mL. TM The therapeutic biologic is analyzed by imaging. Qualitatively, the SMP is in a range comparable to the starting monomeric therapeutic biologic solution. In certain embodiments, the composition is substantially free of submicron particles (SMP) when dissolved in an aqueous liquid.

[0144] As described herein, the present disclosure relates to a highly concentrated composition comprising a flocculant and a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein the particles (e.g., substantially all of the particles) comprise at least one therapeutic biologic, wherein when dissolved in water, a buffer or other physiologically relevant aqueous liquid (e.g., a biological fluid in a patient), the immunogenicity of the composition is substantially similar compared to a similar aqueous composition comprising a monomeric therapeutic biologic. As used herein, "physiologically relevant" conditions that may be encountered in a mammal or human body can be applied. Those skilled in the art will be able to determine the most suitable set of conditions for testing based on the final application of the composition described herein. In some embodiments, when dissolved in an aqueous liquid, the immunogenicity of the composition is substantially similar compared to an aqueous composition comprising at least one therapeutic biologic in a soluble form (e.g., a monomeric form). In specific embodiments, the composition is substantially non-immunogenic. In certain embodiments, the aqueous liquid is water, an aqueous buffer or a physiologically relevant aqueous liquid.

[0145] As disclosed herein, the term "immunogenicity" refers to the induction of an immune response by a composition of an injected therapeutic biologic (antigen), while "antigenicity" refers to the reaction of a composition of a therapeutic biologic with pre-existing antibodies. In general, antigenicity and immunogenicity are referred to as "immunoreactivity". In particular embodiments, the immunogenicity of the composition is substantially similar to that of an aqueous composition comprising at least one therapeutic biologic in a soluble form (e.g., monomeric form). In certain embodiments, for example, the composition is substantially non-immunogenic when subjected to the presence of a non-immune response based on a repeated dose anti-drug antibody immunogenicity analysis (see, e.g., Example 8).

[0146] The ratio between the toxicity and therapeutic effect of a particular composition (e.g., human IgG) is its therapeutic index and can be expressed as LD 50 (the amount of compound that is lethal to 50% of the population) and ED 50 The ratio between (the amount of compound effective for 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 to formulate a range of doses for use in humans and known in the art. Doses of such compounds are preferably in the range that includes the ED 50The dosage may vary within this range depending on the dosage form employed and the route of administration utilized (e.g., subcutaneous injection). See, e.g., Fingl et al., THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Chapter 1, page 1, 1975. The exact composition, route of administration, and dosage may be selected by the individual physician depending on the patient's condition and the particular method of delivering the composition.

[0147] In some embodiments, the toxicity of the composition is substantially similar to that of an aqueous composition comprising at least one therapeutic biologic in soluble form (e.g., monomeric form). In some embodiments, the toxicity of the composition is reduced compared to an aqueous composition comprising at least one therapeutic biologic in soluble form (e.g., monomeric form). In particular embodiments, the composition is substantially non-toxic, e.g., based on local tolerance and clinical observation analysis (see, e.g., Example 7).

[0148] In certain embodiments according to the present disclosure as described herein, the stability of the therapeutic biologic of the particulate composition is improved compared to an aqueous composition comprising at least one therapeutic biologic in a soluble form (e.g., a monomeric form) (see e.g. Figure 3). A "stable" composition refers to a composition wherein all therapeutic biologics are stored (e.g., in a container closure) at an expected storage temperature (e.g., 4-40°C) and substantially maintain their physical stability and / or chemical stability and / or biological activity. It is desirable that the composition substantially maintains its physical and chemical stability and biological activity during storage. The storage period is generally selected based on the expected shelf life of the composition. In addition, after the composition is frozen (to, e.g., -70°C) and thawed, e.g., after 1, 2, or 3 cycles of freezing and thawing, the composition should be stable. Various analytical techniques for measuring protein stability are available in the prior art and are reviewed, for example, in: Peptide and Protein Drug Delivery, 247-301, Vincent Lee, ed., Marcel Dekker, Inc., New York, NY. (1991) and Jones, A. Advanced Drug Delivery Review. 10: 29-90 (1993). Stability can be measured at a selected temperature over a selected period of time. Stability can be qualitatively and / or quantitatively assessed in a variety of different ways, including assessment of aggregate formation (e.g., using size exclusion chromatography and / or by visual inspection); assessment of charge heterogeneity by using cation exchange chromatography or capillary zone electrophoresis; amino-terminal or carboxyl-terminal sequence analysis; mass spectrometry; SDS-PAGE analysis for comparison of reduced and intact antibodies; peptide mapping (e.g., trypsin or LYS-C) analysis; assessment of the biological activity or antigen binding function of the antibody, etc. 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 three months. In certain embodiments, the therapeutic biologic in the composition is stable for at least one month at 40°C.

[0149] In certain embodiments, the method disclosed herein further comprises administering at least one hyaluronic acid degradation agent of a pharmaceutically effective amount, and the at least one hyaluronic acid degradation agent can be administered simultaneously, sequentially or intermittently with the composition. The subcutaneous tissue or extracellular matrix comprises a network of fibrin in the viscoelastic gel embedded in glycosaminoglycans. Hyaluronic acid is an important glycosaminoglycan in the subcutaneous tissue. Hyaluronic acid is secreted into the stroma by fibroblasts as a viscous polymer and subsequently locally degraded in the lymph by the action of hyaluronidase. Glycosaminoglycans are complex linear polysaccharides of the extracellular matrix, and are characterized in that the repeating disaccharide structure of the hexosamine and uronic acid substituted by N, such as the case of hyaluronic acid.

[0150] Hyaluronic acid degradation agent can strengthen the subcutaneous administration of the composition comprising multiple particles and flocculants, for example, by strengthening and / or increasing the volume of the composition administered by injection, thus improving the absorption of treatment biologicals.Using hyaluronic acid degradation agent (such as hyaluronidase) can improve the subcutaneous administration of treatment biologicals entering systemic circulation by the reversible hydrolysis of hyaluronic acid (for example, hyaluronic acid reversible degradation).The hyaluronic acid degradation in the extracellular matrix temporarily opens the passage in the subcutaneous tissue, thus allowing larger volume to be safely and comfortably applied to the subcutaneous tissue (see, for example, example 9).In addition, the hyaluronic acid degradation can temporarily reduce the viscosity of the subcutaneous tissue and promote the dispersion of the liquid injected, so as to promote its absorption.The effect of hyaluronidase is local and reversible, and wherein the hyaluronic acid tissue is completely reconstructed within 24 to 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]. Increasing the permeability of subcutaneous tissue by degradation of hyaluronic acid is relevant to the efficacy of hyaluronidase because it can increase the dispersion and absorption of compositions comprising multiple particles that are administered simultaneously, sequentially or intermittently. In certain embodiments, the methods described herein further comprise administering at least one hyaluronic acid degrading agent.

[0151] In certain embodiments, the method of the present disclosure further comprises the use of at least one hyaluronic acid degradation agent, for example, at least one hyaluronic acid degradation agent of a pharmaceutically effective amount simultaneously, sequentially or intermittently applied with the composition. In certain embodiments, the hyaluronic acid degradation agent is a hyaluronidase (hyaluronidase enzyme), also referred to herein as a hyaluronidase (hyaluronidase). In certain embodiments, the hyaluronidase is a soluble neutral active hyaluronidase. In certain embodiments, the hyaluronidase is a mammalian hyaluronidase. In a specific embodiment, the mammalian hyaluronidase is a human hyaluronidase. In a specific embodiment, the human hyaluronidase is a recombinant human hyaluronidase, for example, rHuPH20. Some recombinant human hyaluronidase suitable for compositions disclosed herein is commercially available, for example, from the rHuPH20 of Halozyme Therapeutics (San Diego, CA) in San Diego, California.

[0152] According to certain embodiments of the present disclosure, the hyaluronic acid degradation agent can be used simultaneously, sequentially or intermittently with the composition. As used herein, the term "simultaneous" or "nearly simultaneous" application refers to the components applied simultaneously, for example, the composition and the hyaluronic acid degradation agent are applied simultaneously. In certain embodiments, the hyaluronic acid degradation agent and the composition are applied with a single composition (for example, co-formulated). In certain embodiments, the hyaluronic acid degradation agent and the composition are applied as separate formulations. However, a component can be applied within a few minutes or hours, for example, at the same medical appointment or doctor's consultation. Such application is referred to as "sequential" application. In certain embodiments, sequential application refers to the sequential application of hyaluronic acid degradation agent and the composition as described herein. In a specific embodiment, the hyaluronic acid degradation agent is first applied, followed by the application of a dosage or multiple dosages of the composition. In certain embodiments, the component can be applied intermittently as a co-formulation or a separate formulation. In a specific embodiment, the hyaluronic acid degradation agent is first applied.

[0153] Methods of the present disclosure

[0154] When carrying out clinical application of therapeutic compositions comprising therapeutic biological products (e.g., mAbs), it will generally be beneficial to prepare a drug or therapeutic composition suitable for the intended application. In certain embodiments, the pharmaceutical composition may contain, for example, at least about 0.1% of an active therapeutic biological product. In some embodiments, the active therapeutic biological product may account for about 2% to about 99% of the unit weight or, for example, about 50% to about 99%, and any range that may be derived therefrom.

[0155] 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. CD20 can be a useful target for B cell lymphomas because this antigen is expressed at very high density on the surface of malignant B cells (i.e., B cells, in which unabated proliferation can lead to B cell lymphomas). The U.S. Food and Drug Administration (FDA) has approved the anti-CD20 antibody rituximab For the treatment of relapsed and previously treated low-grade non-Hodgkin's lymphoma (NHL). Rituximab works by binding to the CD20 antigen on B cells, which causes B cells to lyse through 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 specific embodiments, the antibody is an anti-CD20 antibody.

[0156] Provided herein are compositions and methods for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a pharmaceutically effective amount of a composition comprising: a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein the particles (e.g., substantially all of the particles) comprise at least one therapeutic biologic or a salt thereof; and a flocculant, wherein the concentration of the flocculant in the composition is less than about 50 mg / mL. In some embodiments, the concentration of the therapeutic biologic or a salt thereof in the composition is greater than about 250 mg / mL. In certain embodiments, provided herein are methods for treating cancer, inflammatory diseases, or immune diseases in a subject in need thereof, the method comprising administering to the subject a pharmaceutically effective amount of a composition comprising: a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein the particles (e.g., substantially all of the particles) comprise at least one therapeutic biologic or a salt thereof; and a flocculant, wherein the concentration of the flocculant in the composition is less than about 50 mg / mL; and wherein the concentration of the therapeutic biologic or a salt thereof in the composition is greater than about 250 mg / mL. Also provided herein are methods for administering a pharmaceutically effective composition comprising: a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein the particles (e.g., substantially all of the particles) comprise at least one therapeutic biologic or a salt thereof; and a flocculant, wherein the concentration of the flocculant in the composition is less than about 50 mg / mL; and wherein the concentration of the therapeutic biologic or a salt thereof in the composition is greater than about 250 mg / mL.

[0157] The term "treat" or "treating" or "treatment" generally refers to therapeutic treatment measures, for example, to treat, reverse and / or downregulate a disease or condition. As used herein, "treat" or "treating" means to apply a therapeutic biologic (such as a composition containing any of the antibodies disclosed herein or an antigen-binding fragment thereof) internally or externally to a subject suffering from one or more disease symptoms or suspected of having a disease for which the biologic has therapeutic or preventive 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 symptoms or inhibiting their progression to a clinically measurable degree. The amount of a therapeutic biologic that is effective to alleviate any particular disease symptom may vary depending on factors such as the disease state, age, and weight of the subject or patient, and the ability of the therapeutic biologic to elicit a desired response in the subject and patient. Whether disease symptoms are alleviated can be assessed by any clinical measurement commonly used by a physician or other skilled healthcare provider to assess the severity or progression of symptoms. The term further includes a delay in the development of symptoms associated with a disorder and / or a reduction in the severity of symptoms of such disorders. The term further includes improving existing uncontrolled or unwanted symptoms, preventing additional symptoms, and improving or preventing the potential causes of such symptoms. Therefore, the term means that a beneficial result has been conferred to a human or animal subject suffering from a disorder, disease or symptom or having the possibility of developing such a disorder, disease or symptom. As used herein, "treatment" refers to therapeutic treatment and diagnostic applications because it is applicable to human or veterinary subjects. "Treatment" applicable to human or veterinary subjects covers contact of therapeutic biological products (e.g., antibodies or antigen-binding fragments of the present disclosure) with human or animal subjects. Subjects in need of treatment of cancer include subjects who already have benign, precancerous or non-metastatic tumors, and subjects in need of preventing the occurrence or recurrence of cancer. In some embodiments, the purpose or result of treatment (treating) or treatment (treatment) can be to reduce the number of cancer cells; reduce the size of the primary tumor; inhibit (i.e., to some extent slow down and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., to some extent slow down and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more symptoms associated with the disorder to some extent. In some embodiments, the efficacy of treatment may be measured by assessing duration of survival, time to disease progression, response rate (RR), duration of response, and / or quality of life.As used herein, the term "prevent" or "preventing" or "prevention" refers to any action that inhibits or delays the onset of a disease or condition in a subject in need thereof, including administering to the subject a pharmaceutically effective amount of a composition according to the present disclosure. In certain embodiments of the present disclosure, "treating" a subject with a disease or condition should include, but is not limited to: (i) slowing, stopping or reversing the progression of the disease or condition, (ii) slowing, stopping or reversing the progression of the symptoms of the disease or condition, (iii) reducing the likelihood of recurrence of the disease or condition, and / or (iv) reducing the likelihood of recurrence of the symptoms of the disease or condition.

[0158] Non-limiting examples of cancers that can be treated by the compositions and methods described herein include, but are not limited to, any solid or non-solid cancer and / or cancer metastasis, including but not limited to gastrointestinal tumors (colon cancer, rectal cancer, colorectal carcinoma, colorectal cancer, colorectal adenoma, hereditary nonpolyposis type 1, hereditary nonpolyposis type 2, hereditary nonpolyposis type 3, hereditary nonpolyposis type 6; colorectal cancer, hereditary nonpolyposis type 7, small intestine and / or large intestine cancer, esophageal cancer, callosity with esophageal cancer, stomach carcinoma, pancreatic cancer, pancreatic endocrine tumors), endometrial cancer, dermatofibrosarcoma protuberans, gallbladder cancer, biliary tract tumors, prostate cancer, prostate adenocarcinoma, kidney cancer (e.g., Wilms' tumor type 2 or type 1), liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma), carcinoma, hepatocellular cancer), bladder cancer, embryonal rhabdomyosarcoma, germ cell tumor, trophoblastic tumor, testicular germ cell tumor, ovarian immature teratoma, uterine, ovarian epithelial, sacrococcygeal tumor, choriocarcinoma, placental site trophoblastic tumor, epithelial adult tumor, ovarian cancer, serous ovarian cancer, ovarian sex cord tumor, cervical cancer, cervical cancer, small cell and non-small cell lung cancer, nasopharyngeal, breast carcinoma (e.g., ductal breast cancer, invasive intraductal breast cancer, sporadic; breast cancer, breast cancer predisposition, type 4 breast cancer, type 1 breast cancer, type 3 breast cancer; breast-ovarian cancer), squamous cell carcinoma (e.g., in the head and neck), neurogenic tumors, astrocytoma, ganglioneuroma, neuroblastoma, lymphoma (e.g., Hodgkin's disease, disease), non-Hodgkin's lymphoma, B cell, Burkitt's, skin T cell, histiocytic, lymphoblastic, T cell, thymus, B cell non-Hodgkin's lymphoma), glioma, adenocarcinoma, adrenal tumor, hereditary adrenocortical carcinoma, brain malignancies (tumors), various other cancers (e.g., bronchogenic giant cell, duct, malignant ascites, Ehrlich-Lettreascites, epidermoid, large cell, Lewis lung, medullary, mucoepidermoid, oat cell, small cell, spindle cell, prickle cell, transitional cell, undifferentiated, carcinosarcoma, choriocarcinoma, cystadenocarcinoma), ependymoma, epithelioma, erythroleukemia (e.g., Friend, lymphoblastic), fibrosarcoma, giant cell tumor, glioma, glioblastoma (e.g.,pleomorphic cell tumor, astrocytoma), glioma, hepatocellular carcinoma, hybridoma, heteromyeloma, histiocytoma, hybridoma (e.g., B cell), adrenal tumor, insulinoma, islet tumor, keratoma, leiomyoma, leiomyosarcoma, leukemia (e.g., acute lymphocytic, acute lymphocytic pre-B cell, acute lymphocytic T cell leukemia, acute megakaryocytic, monocytic, acute myeloid, acute myeloid like, acute myeloid with eosinophilia, B cell, basophilic, chronic myeloid, chronic, B cell, eosinophilic, Friend, granulocytic or myeloid, hairy cell, lymphocytic, megakaryocyte, monocytic, monocytic-macrophage, myeloblastic, myeloid, myelomonocytic, plasma cell, pre-B cell, promyelocytic, subacute, T cell, lymphoid neoplasm, myeloid malignancy susceptibility, acute nonlymphocytic leukemias), lymphosarcoma, melanoma, breast tumors, mastocytoma, medulloblastoma, mesothelioma, metastatic tumors, mononuclear cell tumors, multiple myeloma, myelodysplastic syndrome, myeloma, nephroblastoma, neural glial tumors, neural neuronal tumors, schwannoma, neuroblastoma, oligodendroglioma, osteochondroma, osteomyeloma, osteosarcoma (e.g., Ewing's), osteoporosis, bone metastasis, papilloma, transitional cell, pheochromocytoma, pituitary tumor (diffuse), plasmacytoma, retinoblastoma, rhabdomyosarcoma, sarcomas (e.g., Ewing's, histiocytic, Jensen's, osteoblastic, reticulocyte), schwannoma, subcutaneous tumors, teratocarcinomas (e.g., pluripotent), teratomas, testicular tumors, thymoma and trichoepithelioma, gastric cancer (gastric cancer), fibrosarcoma, glioblastoma multiforme; multiple glomus tumors, Li-Fraumeni syndrome, liposarcoma, lynch cancer family syndrome II, male germ cell tumor, mast cell leukemia, medullary thyroid, multiple meningiomas, endocrine neoplasia myxosarcoma, paraganglioma, familial non-chromaffin, calcifying epithelioma, papillary, familial and sporadic, rhabdoid susceptibility syndrome, familial, rhabdoid tumor, soft tissue sarcoma, or Turcot syndrome with glioblastoma.

[0159] 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. In specific embodiments of the compositions and methods described herein, it can be used to treat non-Hodgkin's lymphoma (NHL) in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of a composition comprising: a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein the particles (e.g., substantially all of the particles) comprise at least one therapeutic biologic or a salt thereof; and a flocculant, wherein the concentration of the flocculant in the composition is less than about 50 mg / mL; and wherein the concentration of the therapeutic biologic or a salt thereof in the composition is greater than about 250 mg / mL. As described herein, any antibody capable of binding to the CD20 antigen can be used in the methods of the present disclosure. Antibodies that bind to the CD20 antigen include, for example: C2B8 (rituximab; ) (U.S. Pat. No. 5,736,137, expressly incorporated herein by reference); yttrium-

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

[0160] In certain embodiments of the present disclosure, the anti-CD20 antibody is rituximab. Rituximab is a genetically engineered chimeric murine / human monoclonal antibody. Rituximab is an IgG, kappa immunoglobulin containing murine light and heavy chain variable region sequences and human constant region sequences. Rituximab has a binding affinity of about 8.0 nM for the CD20 antigen and is commercially available, for example, from Genentech (South San Francisco, CA) in South San Francisco, California.

[0161] In other embodiments, the antibody is trastuzumab (Herceptin; Genentech, South San Francisco, CA). Trastuzumab is a humanized monoclonal antibody that binds to the extracellular portion of the HER2 extracellular domain, preventing dimerization and the cascade that leads to growth factor expression. In certain embodiments, trastuzumab is used to treat breast cancer, esophageal cancer, gastric cancer, or other new or metastatic cancers that overexpress HER2.

[0162] Also provided herein are methods for treating an inflammatory disease in a subject in need thereof, the method comprising administering to the subject a pharmaceutically effective amount of a composition comprising: a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein the particles (e.g., substantially all of the particles) comprise at least one therapeutic biologic or a salt thereof; and a flocculant, wherein the concentration of the flocculant in the composition is less than about 50 mg / mL; and wherein the concentration of the therapeutic biologic or a salt thereof in the composition is greater than about 250 mg / mL.

[0163] In some embodiments of the methods described herein, inflammatory diseases include, but are not limited to, joint diseases, ophthalmic diseases, retinal diseases, Crohn's disease, irritable bowel syndrome, 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, pneumonia, encephalitis, meningitis, myocarditis, osteomyelitis, myositis, hepatitis, gastritis, enteritis, appendicitis, pancreatitis, or cholecystitis.

[0164] In some embodiments, the compositions and methods described herein can be used to treat immune diseases, such as acquired hypogammaglobulinemia secondary to hematological malignancies, chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barre Syndrome, idiopathic thrombocytopenic purpura, inflammatory myopathy, Lambert-Eaton myasthenic syndrome, multifocal motor neuropathy, myasthenia gravis, Moersch-Woltmann syndrome, secondary hypogammaglobulinemia specific antibody deficiency, acute disseminated encephalomyelitis, autoimmune hemolytic anemia; cicatricial pemphigoid, Evans syndrome; syndrome), fetal-maternal / neonatal alloimmune thrombocytopenia (FMAIT / NAIT), hemophagocytic syndrome, high-risk allogeneic hematopoietic stem cell transplantation, IgM paraproteinemia neuropathy, renal transplantation, multiple sclerosis, opsoclonus-myoclonus ataxia, post-transfusion purpura, toxic epidermal necrolysis / Steven Johnson syndrome (Toxic epidermal necrolysis / Steven Johnson syndrome, TEN / SJS), toxic shock syndrome, Alzheimer's disease, multiple myeloma, sepsis; B cell tumors, trauma or bacterial, viral, fungal infection. In some embodiments, the immune disease is an autoimmune disease. In certain embodiments, autoimmune diseases include, but are not limited to, multiple sclerosis, scleroderma, type I diabetes, rheumatoid arthritis, thyroiditis, Reynaud's syndrome, Sjögren's syndrome, autoimmune uveitis, autoimmune myocarditis, inflammatory bowel disease, amyotrophic lateral sclerosis (ALS), systemic lupus erythematosus, neuromyelitis optica, idiopathic thrombocytopenic purpura, myasthenia gravis, ulcerative colitis, Crohn's disease, polyarthritis, graft-versus-host reaction, juvenile-onset diabetes, Hashimoto's thyroiditis, Grave's disease, pernicious anemia, chronic active (lupus-like) 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 (e.g., such as focal segmental glomerulosclerosis (FSGS)) comprises reducing the likelihood of developing the disease by at least about 20%, such as by at least about 30%, 40%, 50%, 60%, 70%, 80%, or 90%.In some embodiments, reducing the risk of a disease comprises a delay in the development of the disease, such as a delay of at least about six months, such as about one year, such as about two years, about five years, or about ten years.

[0165] As described herein, by achieving injectability with low injection force, without the need for resuspending before injection, a particle-based composition with a flocculant can directly address the current challenge of subcutaneous administration of therapeutic biologics (e.g., mAbs). In some embodiments, particles and flocculants are suspended in a non-aqueous liquid carrier and remain substantially suspended in the liquid carrier for at least one month, thereby eliminating the need for complex, time-consuming resuspension procedures. The compositions described herein can be used for prefilled syringes, prefilled portable drug delivery injection devices, or prefilled oral administration liquid syringe capsules. The highly dispersible nature of the particles in the compositions described herein allows patient-friendly subcutaneous injections. In the subcutaneous space, as described herein, the therapeutic biologics contained in the particles are easily restored to their original monomeric state after injection, thereby achieving complete bioavailability. In a specific embodiment, the composition does not damage the quality of the therapeutic biologics and achieves a higher load, thereby allowing the therapeutic biologics (e.g., mAbs) to be easily delivered by subcutaneous injection to treat a disease or condition of a subject in need, wherein the disease or condition is cancer, inflammatory disease, or 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.

[0166] Pharmaceutical compositions and methods as disclosed herein can be administered to a subject by any suitable route of administration, including, for example, parenteral or intraperitoneal. In some embodiments, the composition is administered by parenteral, subcutaneous, oral, epidermal, intradermal, intramuscular, intraarterial, intraperitoneal or intravenous injection. In a particular embodiment, the composition is administered by subcutaneous injection. The details of the appropriate route of administration and the composition suitable for the route of administration can be found, for example, in the following: U.S. Patent No. 6,110,973; No. 5,763,493; No. 5,731,000; No. 5,541,231; No. 5,427,798; No. 5,358,970 and No. 4,172,896 and patents cited therein. The term "pharmaceutical composition" as disclosed herein refers to a preparation whose form allows the therapeutic biological product in the composition to be effective (e.g., when administered to a subject), and which does not contain additional components that are unacceptably toxic to the subject to whom the composition is administered. Such compositions are sterile. A "sterile" composition or formulation is sterile or free of all living microorganisms and their spores.

[0167] The term "injectability" or "syringeability" refers to the relative ease with which a liquid composition as described herein can be administered to a subject using an injection device (e.g., a syringe, a portable drug delivery injection device, or an oral liquid syringe capsule). Specifically, injectability is partially affected by the viscosity of the composition, and more importantly, by the sedimentation, injection or transfer flow rate of the particles, and needle characteristics (e.g., length and gauge). In some embodiments, injectability is determined by measuring the viscosity of the composition at various shear rates. In some embodiments, injectability is determined by measuring the separation force and / or sliding force required to actuate an injection device consisting of a barrel, a plunger, and a needle. In certain embodiments, the inner diameter of the barrel of the syringe is at least 6 mm. As described herein, the injectability of a composition comprising a flocculant and a plurality of particles comprising at least one therapeutic biological product is superior to the injectability of an aqueous formulation of an aqueous monomer therapeutic biological product having approximately the same concentration. The term "injectability" or "syringeability" may also refer to the injectability of a pharmaceutical composition through a syringe equipped with a 16-33 gauge needle (optionally, a thin-wall or ultra-thin-wall (UTW) needle). In certain embodiments, the syringe is equipped with a needle having a length of at least 8 mm. Injectability depends on factors such as the pressure or force required for injection, flow uniformity, quality of aspiration, and lack of clogging. As described herein, injectability can be assessed by comparing the injection force of the composition after a certain period of time with a standard granular composition to which no flocculant is added. When compared to a standard granular composition of a therapeutic biologic having the same concentration under otherwise identical conditions, a composition as described herein can improve injectability after a certain period of time, wherein the injection force is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, and most preferably by at least 75%. Alternatively, the injectability of the composition can be evaluated by comparing the time or injection force required to inject the flocculated volume (such as injecting 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 over a certain period of time when the syringe is pressed down with the same force), for example, the sedimentation of particles over a certain period of time. The phrase "flow rate" is the volume of a liquid composition that can pass through a given cross-sectional area per unit time. Generally, the flow rate formula is Q=A×v, where Q is the flow rate, A is the cross-sectional area at a point on the flow path, and v is the average velocity of the liquid at that point. In certain embodiments, the flow rate is constant. In specific embodiments, the flow rate is at least about 0.1 ml / sec.

[0168] The term "injection separation force" refers to the force required to overcome the friction between the syringe barrel and the plunger of a standard injection device before the contents of the syringe can be ejected at a stable rate, for example, the maximum force required to break the static friction of the plunger. The force is applied at the outward end of the syringe plunger shaft and is directed along the axis of the syringe barrel. The contents of the syringe are ejected through a syringe needle of a specified gauge and length. In certain embodiments, the injection separation force is measured during actuation by a load cell placed at the outward end of the syringe plunger.

[0169] The terms "syringe force", "injection force", "injection sliding force" or "sliding force" are used interchangeably herein and refer to the force required to maintain a stable ejection of the contents of a standard injection device, e.g., once static friction is overcome, the force required to maintain the plunger movement. The force is applied to the outward end of the syringe plunger shaft and is oriented along the axis of the syringe barrel. The contents of the syringe are ejected through a syringe needle of a specified size and length. The term "Newtonian system" or "N" means a range of shear stresses that is linearly proportional or nearly linearly proportional to the local strain rate at each point. In some embodiments, adding a flocculant to a suspension of particles reduces the syringe force (see, e.g., Examples 16 and 17).

[0170] The administration of the compositions described herein may comprise administration using a 18-33 gauge needle. The length of a 18-33 gauge needle may be about 19 mm (3 / 4 inch) or less; or preferably about 13 mm (1 / 2 inch) or less. In some embodiments, the administration of the compositions uses a 27-33 gauge needle. In some embodiments, the length of a 27-33 gauge needle is about 13 mm (1 / 2 inch) or less. In certain embodiments, the compositions are dispensed from a needle having a gauge range of 18 to 33 (e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 to 33). In some embodiments, the compositions are dispensed from a needle having a gauge range of 27 to 30. In certain embodiments, the compositions are dispensed from a needle having a gauge range of 25 to 27. In a particular embodiment, the compositions are dispensed from a needle having a gauge of 27.

[0171] In some embodiments, the composition is dispensed using an injection force of less than about 70N, for example, about 60, 50, 40, 30, 25, 20, 15, 10 or 5N. In some embodiments, the composition is dispensed using an injection force of less than about 25N. In a particular embodiment, the composition is dispensed using an injection force of less than about 20N. In certain embodiments, the composition is dispensed using an injection force of less than about 15N. In some embodiments, the composition is dispensed using an injection force of less than about 10N. In certain embodiments, the composition is dispensed using an injection force of less than about 5N. In some embodiments, as the concentration of the therapeutic biologic in the composition increases, the injection force increases at a rate lower than the viscosity of the composition. In certain embodiments, the injection force remains substantially the same for at least one month under container-closed storage conditions of less than about 40°C.

[0172] In other cases, the composition comprising multiple particles and flocculants as described herein, optionally, further comprises the use of at least one hyaluronic acid degradation agent (for example, hyaluronidase), which is used simultaneously, sequentially or intermittently with the composition. In certain embodiments, the composition to be applied is less than about 20.0mL, for example, 15.0, 10.0, 5.0, 2.0, 1.5, 1.0, 0.5mL. In certain embodiments, the composition to be applied is less than about 2.0mL. In certain embodiments, the composition to be applied is less than about 1.5mL. In certain embodiments, the composition to be applied is less than about 1.0mL. In a specific embodiment, the composition to be applied is less than about 0.5mL.

[0173] 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, for example, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49 ... 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 seconds. In specific embodiments, the composition dissolves immediately after administration.

[0174] Compositions described herein can be used for parenteral administration, for example, formulated for injection by intravenous, intramuscular, subcutaneous or even intraperitoneal routes, without the need for manual stirring or premixing of injection devices before use. Pharmaceutical compositions suitable for injectable use include sterile non-aqueous liquid carriers comprising sterile particles. In all cases, the composition must be sterile and must be a fluid that can be easily injected. The pharmaceutical composition should also be stable under current good manufacturing procedures (cGMP) and storage conditions.

[0175] The present disclosure generally relates to compositions for syringes or portable drug delivery injection devices. In certain embodiments, the composition is located in a syringe or a portable drug delivery injection device. For example, the syringe disclosed herein can be a prefilled syringe. In certain embodiments, the syringe or portable drug delivery injection device is prefilled with the composition. The example of a portable drug delivery injection device includes a pen-type syringe, an automatic injector (automatic injector) (e.g., an automatic injector (autoinjector), a patch) or an oral administration liquid injector capsule (e.g., a robot pill for delivering drugs in the gastrointestinal tract, etc.). The compositions described herein can be enclosed in a disposable syringe, a cartridge or any other container closure made of glass or plastic, etc. In certain embodiments, the composition is administered by injection of a syringe. In certain embodiments, the composition is dispensed from a prefilled syringe. In certain embodiments, the portable drug delivery injection device is configured to automatically or semi-automatically deliver the drug to the patient using a wireless communication system.

[0176] An automatic injector, such as an autoinjector, a pen injector or a drug pen or an oral administration liquid injector capsule, is a prefilled and preassembled injection device that enables parenteral administration of a therapeutic agent. The advantage of an automatic injector is that it contains a measured dose of a therapeutic agent in a sealed sterile cartridge or injection device. An automatic injector can be used for people with low dexterity, poor eyesight, or who need portability to administer the therapeutic agent on time, and can also reduce the fear or adversity of self-injection of the therapeutic agent, which increases the likelihood that the person will take the drug. An automatic injector is most useful in emergency situations, where it can quickly and simply administer the therapeutic agent by itself without measuring the dose.

[0177] Ensuring complete delivery of the particle suspension of the therapeutic biologic in the compositions and methods disclosed herein is often important for particle suspensions for which injection forces may already be relatively high. Under high shear, as is the case with portable drug delivery injection devices, the problem of ensuring complete injection of a unit dose under high injection forces may be further complicated by particle settling in the composition, making operation challenging for the user.

[0178] In an automatic injector, the particles of the therapeutic biologic in the composition may be stored as a particle suspension and subsequently injected. However, long-term storage of particle suspensions (e.g., 3 to 12 months) has disadvantages, such as particle sedimentation, which may result in high injection forces. These automatic injectors require the user to manually shake the syringe body immediately before injection to speed up the resuspension of the particles. Unfortunately, steps such as manually shaking the automatic injector 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.

[0179] In some embodiments, the composition is administered by a syringe or a portable drug delivery injection device. In a specific embodiment, the composition is located in a syringe or a portable drug delivery injection device. In a specific embodiment, 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-type syringe. In certain embodiments, the portable drug delivery injection device is an automatic syringe. In certain embodiments, the portable drug delivery injection device is an oral administration liquid syringe capsule. In a specific embodiment, the syringe or portable drug delivery injection device is pre-filled with the composition. In certain embodiments, the composition is administered in one or more doses. In certain embodiments, the composition is administered in a single dose. In certain embodiments, the composition is administered in multiple doses.

[0180] 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 autoinjector. In some embodiments, the composition is administered by an oral administration liquid injector capsule. In a specific embodiment, the syringe, pen injector, autoinjector, oral administration liquid injector capsule is prefilled.

[0181] The compositions of the present disclosure can be used to treat a disease or condition in a subject in need. 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, for example, a human or an animal. Compositions as described herein can be administered to a mammal, for example, a human or an animal subject, in vivo using various known approaches and techniques. For example, the composition can be provided as an injectable suspension and administered by parenteral, subcutaneous, oral, epidermal, intradermal, intramuscular, intraarterial, intraperitoneal or intravenous injection using a conventional needle and syringe or a portable drug delivery injection device. In certain embodiments, the composition is administered to a mammal. In a specific embodiment, the composition is administered to a human.

[0182] In certain embodiments according to the present disclosure, when the composition is administered, it can be completely dissolved immediately or within a few seconds, thereby mitigating any immune risk caused by particles that persist in the subcutaneous space. For example, administration of particles comprising mAbs has been shown to produce similar or improved pharmacokinetic (PK) curves (AUC, Cmax, and Tmax) as described herein compared to an equal dose of aqueous monomeric mAbs. In certain embodiments, the composition dissolves immediately after administration.

[0183] Compositions described herein demonstrate subcutaneous delivery of high concentrations of therapeutic biologics (300-750 mg / mL) compositions that do not lose biological activity. Various therapeutic biologics have achieved this by using organic solvents, such as sesame oil, medium chain triglycerides (MCT), saturated vegetable fatty acids C8 and C10 propylene glycol diesters (PGD), fatty acid esters (ethyl oleate (EO)) as liquid carriers. For example, ethyl oleate is a fatty acid ester with a viscosity of about 6 mPa s at 25 ° C, and does not chemically interact with particles during storage or dissolution in vivo.

[0184] Dense (1.32 g / cm2) particles with controlled size distribution (polydispersity index <0.2) can be achieved using various therapeutic biologics described herein. 3) round particles. Focused ion beam scanning electron microscopy (FIB-SEM) can be used to determine whether the particles are free of void space, which is critical for achieving 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 for achieving low viscosity compositions, but small enough to prevent syringe clogging in a 27-gauge needle. For example, by loading particles having a flocculant and a protein concentration of about 400 mg / mL (in an ethyl oleate (EO) liquid carrier) into a pre-filled syringe, a therapeutic composition having a viscosity of about 20 mPa·s (associated with an injection force of about 4 N) can be formed, which can be stored for at least one month without substantial changes 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 flocculation volume of the composition is substantially the same over at least one month. See Example 6. In some embodiments, a plurality of particles and flocculants 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 a flocculant to the composition can prevent particle settling and prevent needle clogging.

[0185] As described herein, characterization of particle structure stability in the composition was accomplished using size exclusion chromatography (SEC), differential scanning fluorimetry (DSF), circular dichroism (CD), cation exchange chromatography (CIEX), and sub-visible particles (SvP) analysis. Additionally, preservation of biological activity was analyzed using pharmacokinetic (PK) and immunogenicity studies. See Examples 7 and 8.

[0186] Also described herein is SEC data demonstrating that minimal aggregate formation was observed upon processing compared to a labeled formulation (an aqueous mAb that is an FDA approved formulation) containing aggregates compared to a reconstituted particle composition containing 96.6% monomer (protein content 507 mg / mL). See Example 11. After 30 days of storage at 40°C, DSF showed that the thermal drift of the samples was less than 1°C, and CD could not detect any differences in secondary structure (percent beta sheets). CIEX was used to analyze charged variants of proteins as specified (ICH Q6B) to ensure that no chemical modifications occurred during the preparation described herein and upon storage. In certain embodiments, the compositions may be less prone to chemical modifications upon storage than FDA labeled formulations. This is due to the protein being more stable as particles in the solid state.

[0187] After storage at 40°C for 30 days, preservation of biological activity was demonstrated by flow cytometry assay. There were no obvious discernible differences between the FDA labeled formulations and those compositions as described herein. As shown in Examples 4 and 5 herein, in the case of the FDA labeled formulations, biological activity decreased significantly after storage, whereas no decrease in activity was observed for the particles in the composition.

[0188] Before the in vivo study began, the particles used for the composition were obtained under essentially sterile conditions. The bacterial endotoxin level was about 3 orders of magnitude lower than the recognized injection standard (0.05 EU / mg to 0.25 EU / mg). In addition, the microbial growth assay indicated no observable growth.

[0189] 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 curves are shown in Example 7, and mAb microparticle suspensions (SC injections) show higher bioavailability than aqueous mAb SC injections. See Fig.11 The in vivo dissolution behavior of the particles has been demonstrated to ensure that the composition can be cleared from the injection site at an increased rate compared to standard aqueous formulations, as undissolved particles may elicit an immunogenic response. In some embodiments, the pharmacokinetics (PK) of the composition is improved compared to aqueous compositions comprising at least one therapeutic biologic in soluble form (e.g., monomeric form). See Fig.11 . The area under the curve (AUC) is from time zero to infinity and represents the total drug exposure over time. Peak concentration is a pharmacokinetic measurement used to determine drug administration. The maximum concentration (Cmax) is the highest concentration of the drug in the blood, cerebrospinal fluid, or target organ after a dose is given. Tmax is the time required for the drug to reach the maximum concentration (Cmax) after administration of a drug that needs to be absorbed (e.g., an oral drug). Tmax is determined by the rate of drug absorption and drug elimination. At Tmax, these values ​​are equal. In certain embodiments, the AUC, Cmax, and / or Tmax of the composition are improved compared to an aqueous composition comprising at least one therapeutic biologic in a soluble form.

[0190] In some embodiments, administration of hyaluronidase allows for increased volume and fluid dispersion of compositions with flocculants administered by subcutaneous syringe injection. As disclosed herein, by pre-injection of hyaluronidase, the injection volume of subcutaneously administered compositions without resuspension can be greatly increased. As shown in Example 9, these hyaluronidase pre-injection sites showed an increase in the injection rate of the composition, as determined by the appearance of skin blisters formed at the application site upon injection.

[0191] Specific embodiments of the present disclosure are described herein. Of course, for those of ordinary skill in the art, after reading the foregoing description, changes, modifications and substitutions of the equivalents of these specific embodiments will become apparent. The inventors expect that the technicians can adopt such changes, changes, modifications and substitutions of equivalents when appropriate, and the inventors intend to make the present disclosure practiced in other ways different from the ways specifically described herein. Those skilled in the art will readily recognize that a variety of non-critical parameters that can be changed, altered or modified to produce essentially similar results. Therefore, where applicable law permits, the present disclosure includes all modifications and equivalents of the subject matter narrated in the attached claims. In addition, unless otherwise specified herein or otherwise clearly contradicted with the context, the present disclosure encompasses any combination of the above-mentioned elements in all possible variations thereof.

[0192] Having generally described the present disclosure, the present disclosure will be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain aspects and embodiments of the present disclosure and are not intended to be limiting.

[0193] Reagent test kit

[0194] In various embodiments, a kit is envisioned, comprising a pharmaceutically effective composition comprising a plurality of therapeutic particles, wherein a flocculant is suspended in a pharmaceutically acceptable liquid carrier. For example, the kit may include one or more additional containers, each container having one or more various materials (such as reagents, optionally in concentrated form and / or devices) that are expected to use the compositions described herein from a commercial and user perspective. Non-limiting examples of such materials include, but are not limited to, diluents, filters, needles, syringes, cartridges, devices, carriers, packaging, containers, vials, and / or tube markings listing contents and / or instructions for use, and package inserts with instructions for use. In some embodiments, the present embodiment contemplates a kit for administering the therapy of the embodiment. The kit may include one or more sealed prefilled syringes, cartridges, or portable drug delivery injection devices containing any of the pharmaceutical compositions disclosed herein. The kit may include, for example, at least a plurality of particles comprising at least one therapeutic biological product, and reagents for preparing, formulating, and / or administering components of the embodiment or performing one or more steps of the disclosed method of treatment. In some embodiments, the test kit may also include a suitable container, which is a container that does not react with the components of the test kit, such as 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 such as plastic or glass. In a particular embodiment, the composition is dispensed from a prefilled syringe or a portable drug delivery injection device. In certain embodiments, the test kit includes a syringe or a portable drug delivery injection device; and a composition comprising: a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein the particles (e.g., substantially all of the particles) include at least one therapeutic biological product or a salt thereof; and a flocculant, wherein the concentration of the flocculant in the composition is less than about 50 mg / mL; and wherein the concentration of the therapeutic biological product or a salt thereof in the composition is greater than about 250 mg / mL.

[0195] The kit may further include instructions outlining the procedural steps of the methods described herein, and will follow substantially the same procedures as described herein or known to those of ordinary skill in the art. The instruction information may be located in a computer-readable medium containing machine-readable instructions that, when executed using a computer, result in the display of a real or virtual procedure for delivering a pharmaceutically effective amount of a therapeutic biologic.

[0196] The mark is optionally located on or associated with the container. For example, when the letters, numbers or other characters forming the mark are attached, molded or etched into the container itself, the mark is located on the container; when the mark is present in a receiver or carrier that also holds the container, for example as a package insert, the mark is associated with the container. In addition, the mark can be used to indicate that the contents will be used for a specific therapeutic application. In addition, the mark also indicates the instructions for use of the contents, such as in the methods described herein. In certain embodiments, the composition is present in a package or dispenser device, which contains one or more unit dosage forms containing the therapeutic biological products provided herein. For example, the package contains metal or plastic foil, such as a blister package. Alternatively, the package or dispenser device can be accompanied by an application instruction. Alternatively, the package or dispenser can be accompanied by a notice associated with the container in a form prescribed by a government agency that controls the manufacture, use or sale of the drug, which reflects the agency's approval of the form of the drug for human or veterinary administration. For example, such notices are prescription drug marks approved by the U.S. Food and Drug Administration, or approved product inserts. In some embodiments, a composition containing the therapeutic particles provided herein and a flocculant is prepared, formulated in a compatible pharmaceutical liquid carrier, placed in an appropriate container, and labeled for treatment of an indicated condition.

[0197] In some cases, the liquid composition as provided herein is deployed in a pre-filled injection device, for example, a syringe or a portable drug delivery injection device. In certain embodiments, a particle suspension (for example, a composition) is deployed as the following volume to be administered: 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 2.5mL. In certain embodiments, the volume of the composition to be administered is less than about 2.5mL. In certain embodiments, the volume of the composition to be administered is less than about 2.0mL, preferably less than about 1.5mL, more preferably less than about 1.0mL, and most preferably less than about 0.5mL.

[0198] In other cases, the method further comprises the use of at least one hyaluronic acid degradation agent, for example, at least one hyaluronic acid degradation agent of pharmaceutically effective amount used simultaneously, sequentially or intermittently with the composition. In certain embodiments, the volume of the hyaluronic acid degradation agent to be applied is less than about 2.0L, for example, less than about 1.8, 1.5, 1.2, 1.0, 0.8, 0.5, 0.3, 0.1L, or 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 2.5mL. In certain embodiments, the composition to be applied is less than about 20.0mL. In certain embodiments, the composition to be applied is less than about 10.0mL. In certain embodiments, the composition to be applied is less than about 5.0mL.

[0199] In some embodiments, such kits include pre-filled injection devices of the present disclosure in blister packs, e.g., syringes or portable drug delivery injection devices. The interior of the blister pack itself can be sterile. In some embodiments, pre-filled injection devices (e.g., syringes or portable drug delivery injection devices) according to the present disclosure can be placed in such blister packs before undergoing sterilization (e.g., terminal disinfection).

[0200] Having generally described the present disclosure, the present disclosure will be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain aspects and embodiments of the present disclosure and are not intended to be limiting.

[0201] Example

[0202] Abbreviations

[0203] Angstrom

[0204] aa amino acid

[0205] BSA bovine serum albumin

[0206] ℃ Celsius

[0207] cm centimeters

[0208] d day

[0209] DI Deionization

[0210] DP dispersed phase

[0211] EA Ethyl acetate

[0212] ELISA enzyme-linked immunosorbent assay

[0213] EO Ethyl Oleate

[0214] eq. equivalent

[0215] Et Ethyl

[0216] eV electron volt

[0217] FDS Filtered Drug Substances

[0218] FA Flocculant

[0219] FV Flocculation volume

[0220] g

[0221] GC Gas chromatography

[0222] h hour

[0223] HDPE High Density Polyethylene

[0224] hIgG Human IgG

[0225] HPLC High Performance Liquid Chromatography

[0226] HSA Human serum albumin

[0227] Hz Hertz

[0228] ID inner diameter

[0229] IV

[0230] K Thousand

[0231] KF Karl Fischer

[0232] kJ Kilojoule

[0233] kPa kilopascal

[0234] kV kilovolt

[0235] L Liter

[0236] LC-MS Liquid chromatography mass spectrometry

[0237] LD laser diffraction

[0238] LDS Liquid Drug Substance

[0239] Lyo Freeze-dried

[0240] m yuan

[0241] mAb monoclonal antibody

[0242] MALDI-MS matrix-assisted laser desorption ionization mass spectrometry

[0243] Me Methyl

[0244] MHz Megahertz

[0245] min

[0246] μg microgram

[0247] μL microliter

[0248] μm micrometer

[0249] μM micromolar

[0250] mg milligram

[0251] mL milliliters

[0252] mm Millimeters

[0253] mM millimole

[0254] mol mole

[0255] mPa·s millipascal·second

[0256] mTorr Millitorr

[0257] N Newton

[0258] nBA n-Butyl acetate

[0259] nm nanometer

[0260] p

[0261] PBS Phosphate buffered saline

[0262] PEG Polyethylene glycol

[0263] PEGA Polyethylene glycol polyacrylamide

[0264] PI Pressure Indicator

[0265] ppm parts per million

[0266] ps picosecond

[0267] PS 80 Polysorbate 80

[0268] PTFE Polytetrafluoroethylene

[0269] rcf relative centrifugal force

[0270] RH Relative humidity

[0271] RP-HPLC Reversed-phase high performance liquid chromatography

[0272] rpm revolutions per minute

[0273] RT Room temperature

[0274] s seconds

[0275] SC Subcutaneous

[0276] sec seconds

[0277] SEM Scanning Electron Microscope

[0278] SLPM Standard liters per minute

[0279] Uncle T

[0280] tert

[0281] UHMW Ultra High Molecular Weight Polyethylene

[0282] um micrometer

[0283] UOM Unit of measurement

[0284] UTW ultra-thin wall

[0285] UV

[0286] V Volts

[0287] VIS Visible

[0288] vol% Volume percentage

[0289] wt% weight percentage

[0290] Material

[0291] Human IgG (IRHUGGF-LY, >97%) and bovine IgG (IRBVGGF) were obtained from Innovative Research as powders or in aqueous solutions. Bovine serum albumin (BSA) and human serum albumin (HSA) were purchased from Sigma-Aldrich. Monoclonal antibodies (mAbs) were provided and received in the form of aqueous solutions. Roche's biosimilar rituximab was purchased from a supplier that provided the antibody in the form of an aqueous composition of 10 mg / mL rituximab, 9 mg / mL sodium chloride, 7.35 mg / mL sodium citrate dihydrate, and 0.7 mg / mL polysorbate 80. Roche's biosimilar trastuzumab was purchased from Genscript Biotech Corporation, which provided the antibody in the form of an aqueous composition. The composition of the custom "feed solution" for processing the particles was generated by modification of the received formulation by desalting, followed by concentration and addition of the desired excipients or by direct buffer exchange. All excipients used in the particle composition have been used in existing approved biologics for injection. Concentration columns were purchased from Millipore Sigma, a manufacturer of proteins for purification and concentration. Ultra 15 mL filter, cut-off 10-50 kDa) and, if necessary, used to: (i) achieve the desired mAb concentration and (ii) exchange buffer / excipients prior to particle formation. In some cases, Zeba desalting columns (THERMO FISHERSCIENTIFIC TM 87773) is also used to remove salt from the solution. Typically, the residual salt to protein ratio (wt / wt) in the desalted solution determined by conductivity measurement and / or elemental analysis is <1%. All excipients were purchased from Sigma-Aldrich and used as received.

[0292] method

[0293] FLOWCAM TM : Using FLOWCAM TM (Dynamic Image Analyzer) measures particle size screening. The sample is diluted to about 1 mg / mL in isopropanol and passed through a thin channel. An image of the particles is recorded and analyzed based on size and shape (count weighted).

[0294] Image analysis: Particle size and circularity were measured using ImageJ analysis on SEM images. Images at, for example, 500x or 1000x magnification were analyzed. The ImageJ particle analysis tool was run on the images to identify objects with circularity>0.8 and size greater than 0.5 μm and the outlines of each object. These outlines were visually inspected to ensure a good fit. Any misidentified particles were manually removed, and any missed particles were manually included and measured using the ImageJ diameter tool. Microscope 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 at least 10 pixels of particle occupancy. This made the particles easy to identify and reduced resolution-based errors. A binary threshold was applied to separate the particles from the background, and a watershed segmentation algorithm was applied to ensure that individual particles were measured separately. The ImageJ tool "Analyze Particles" was then applied to the binary image using the following parameters: circularity of 0.5 to 1.0; size of 5 to infinity square microns; edge exclusion; filling holes. The outlines of the identified particles were overlaid on the original image. Misidentified particles were then discarded, such as clusters identified as single particles or particles whose outlines did not match the particles. Missed particles were measured by manually tracing the outlines of the particles and using the measurement tool of ImageJ.

[0295] Accelerated storage solution : All samples were transferred to glass bottom plates and 2R Schott vials for aging (usually 2 mL or 4 mL volume, depending on the sample). Glass bottom plates and 2R Schott vials were sealed with parafilm, placed in an oven at 4°C, 25°C, 40°C, 50°C, or 60°C, and visually inspected during the aging period to ensure integrity and stability.

[0296] Viscosity measurement Unless otherwise stated, the suspension viscosity was measured at 25°C using an AR-G2 rheometer (TA Instruments) and a 25 mm plate. The measurements were performed at 1000 s-1, which is lower than 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. Prior to each measurement, a calibration standard was recorded to verify the instrument settings.

[0297] Injectability measurement Unless otherwise stated, syringe force (injection force) was measured using a custom-made force sensor device and a 1-mL Normject-type syringe with a 27-gauge ultra-thin-wall needle (TSK Corporation (TSK)) during ejection of 1 mL of a suspension (400 mg / mL particles) at 0.1 mL / sec.

[0298] Karl Fischer: Moisture content testing was performed using Karl Fischer analysis. Approximately 100 mg of the granules were heated to 150° C. in an oven and the released water was determined by coulometry.

[0299] Bone density : Bone density is measured by gas pycnometry. The gas is nitrogen or other compatible gas, and the particle mass is 0.0413 g.

[0300] 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 solution, 96-well plate). After a certain period of time, a 10 μL aliquot was taken from the sample vial and the absorbance at 280 nm was measured and recorded. The Ab (e.g., mAb) concentration versus time for all samples was plotted.

[0301] Salt content : Salt content was recorded by measuring sodium content using inductively coupled plasma optical emission spectroscopy (ICP-OES). Sodium standards ( A calibration curve was prepared using a 100 ppm sodium standard solution (1000 mg / L). Quality control was accomplished using a diluted standard solution of 100 ppm sodium. A sample (about 15 mg) of the particles dissolved in 2 vol% nitric acid (10 mL) was then analyzed, which produced an intensity below the instrument's detection limit of about 0.5 ppm for sodium. This indicates a sodium content of less than 0.034 wt% and a total salt content of less than 0.1 wt% (assuming that sodium citrate and sodium chloride have been removed in equal amounts).

[0302] Size Exclusion Chromatography (SEC) Measurements :AGILENT ADVANCEBIO TM SEC (300 mm × 2.7 μm, Column), 20 μL sample (1 mg / mL) injection was run at a flow rate of 1 mL / min in SEC buffer (25 mM phosphate, 250 mM NaCl, pH 6.8) for 15 minutes. Peak analysis was performed by automatic integration using the following parameters: slope sensitivity = 0.5, peak width value = 0, minimum peak height = 0, minimum peak area value = 0, shoulder peak off, minimum peak area percentage 0, standard tangent cutting mode, advanced baseline correction, front peak cutting height ratio of 0, tail peak cutting height ratio of 0, peak valley ratio of 0, and cutting valley ratio of 0. Alternatively, in AGILENT ADVANCEBIO TM SEC (300 mm × 2.7 μm, Column), in SEC buffer (25mM phosphate, 250mM NaCl, pH 6.8), run 20μL sample (1mg / mL) injection at a flow rate of 1 ml / min for 15 minutes. Use the following parameters for peak analysis by automatic integration: slope sensitivity = 0.5, peak width value = 0, minimum peak height = 0, minimum peak area value = 0, shoulder peak off, minimum peak area percentage 0, standard tangent cutting mode, advanced baseline correction, front peak cutting height ratio is 0, tail peak cutting height ratio is 0, peak valley ratio is 0, and cutting valley ratio is 0.

[0303] Cation Exchange Chromatography (CIEX) Measurement : Under accelerated storage conditions, use AGILENT BIOMAB TM NP5, 4.6×250 mm, PEEK ion exchange column, charge variant analysis was performed on each sample at day 0, day 7, and day 30. Samples were prepared at a concentration of 1 mg / mL after dialysis overnight 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 plus NaCl: 175 mM. Samples were run with a gradient starting with 100% buffer A, ramping to 100% buffer B over the course of 20 minutes, after which the gradient was set to return to 100% buffer A and 0% buffer B over the next 1 minute. The system was re-equilibrated in 100% buffer A for 10 minutes before the next sample was injected. Integration was performed in manual peak skimming mode to reflect the AGILENT TM data.

[0304] Flow cytometry : 1 million Raji cells (100 μL / well) were plated in each well of a 96-well 'V-bottom' plate, and 10 μL of mAb, marker, particle or suspension at a starting concentration of 200 μL was added to the well. The dilution factor of mAb markers, 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 3 times with PBS. 100 μL of PE-conjugated goat anti-human IgG was added as a secondary antibody at a dilution of 1:200. The plate was centrifuged at 2000 pm for 5 minutes and washed 3 times with PBS. The cells were then resuspended in 200 μL of cold PBS to be incubated in Life Technologies ATTUNE TM The cells were analyzed on an NXT flow cytometer.

[0305] Scanning electron microscopy (SEM) : Use HITACHI TMElectron micrographs of selected samples are collected using a TM3030Plus or TM1000 benchtop microscope. Samples are mounted on conductive tape and examined in a low vacuum anti-charging environment, thus avoiding the need for sample preparation.

[0306] Image analysis Microscope 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 a particle occupancy of at least 10 pixels. This made the particles easy to identify and reduced resolution-based errors. A binary threshold was applied to separate the particles from the background, and a watershed segmentation algorithm was applied to ensure that individual particles were measured individually. The ImageJ tool "Analyze Particles" was then applied to the binary images using the following parameters: circularity of 0.5 to 1.0; size of 5 to infinity square microns; edge exclusion; filling of holes. The outlines of the identified particles were overlaid on the original image. Misidentified particles, such as clusters identified as single particles or particles whose outlines did not match the particles, were then discarded. Missed particles were measured by manually tracing the outlines of the particles and using ImageJ's measurement tool.

[0307] Density analysis : By using ACCUPYC TM Approximately 0.1 g of powder was examined by the II 1340 Gas Displacement Pycnometry System to determine the skeletal density of particles from the selected sample.

[0308] Water content analysis : The moisture in the particles from selected samples was determined by placing approximately 0.1 g of the powder in an oven with a Karl Fischer titrator and heating the sample.

[0309] ELISA assay ELISA assay is used to detect human antibodies in a denaturation-sensitive manner on selected samples. First, human IgG is plated in PBS for 1 hour, then washed three times with washing buffer (PBS+0.05% Tween 20) for 4 minutes, then blocked in washing buffer with 2% BSA (Sigma) for 45 minutes, then with diluted (20 μg / mL) protein A-HRP (ABCAM TM ) for 45 min, followed by three incubations with wash buffer for 3 min, followed by TMB (ABCAM TM ) for 10 minutes and then finally with STOP solution (ABCAM TM ) to quench the reaction solution. TM Colorimetric readings were performed on the

[0310] Subvisible Particle (SvP) Analysis: Using Fluid Imaging Technologies FLOWCAM TM The PV-100 system analyzes subvisible particles (SvP). Samples for analysis were mixed in sterile centrifuge tubes with filtered water (MILLI-Q TM ) was reconstituted to the concentration of interest. 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.

[0311] Accelerated storage : Unless otherwise stated, selected samples were stored under accelerated conditions by maintaining them in an incubator or oven at elevated temperature (40°C) for a defined period of time. Samples were stored in 2 mL or 4 mL WHEATON TM glass vials and sealed with parafilm.

[0312] Inverse Gas Chromatography (IGC) : Powdered samples are analyzed using reversed gas chromatography. A cylindrical column is filled with 200 to 300 mg of powdered sample to constitute the stationary phase. After an inert gas purge, a series of gas probes are injected onto the column. Determining the retention volume of each probe allows the evaluation of the dispersion and polar components of the surface energy of each sample.

[0313] X-ray diffraction (XRD) : The samples were packed into 0.7 mm diameter glass capillaries. The powder pattern was obtained on a microscope equipped with an incident beam focusing mirror and X'CELERATOR TM PANALYTICAL EMPYREAN OF DETECTOR TM The patterns were measured using Mo Ká radiation (1-50° 2θ, 0.0167113° step size, 4 sec / step, 1 / 4° divergence slit, 0.02 rad Soller slit). If electrostatic effects (which occur after grinding in a mortar and pestle in the case of evaluating freeze-drying control) prevented the sample from being packed into the capillary, a 100 μm diffractometer was used to measure the sample. TM BRUKER Position Sensitive Detector TM The powder pattern was measured from a flat sample on a D2 phase diffractometer. The pattern was measured with Cu Kα radiation from 5-100° 2θ in steps of 0.0202144°, counting 1.0 sec / step. Standard instrument settings (30 kV, 10 mA, 0.6 mm divergence slit, 2.5° Soller slit, and 3 mm scattering screen height) were used.

[0314] Microfluidic Particle Size Screening (MPS) : Using FLOWCAM TMPV-100 flow imaging microscope for particle size analysis. To study the size and dispersibility of the particles, 5 mg of powder was dispersed in 10 mL of dry isopropanol by ultrasonic treatment. The isopropanol continuous phase prevents the particles from dissolving, i.e., preventing reconstitution. 0.3 mL was injected into the cells and images of the particles were taken using a flow rate of 0.15 ml / min. Particles with a circularity greater than 0.9 were reported in the analysis, and any double images were removed from the analysis to give the size distribution and dispersibility of the particles in the range of 1 to 100 μm.

[0315] Dynamic Vapor Sorption (DVS) : Powders were analyzed using dynamic water vapor adsorption. Approximately 50 mg of powdered sample was loaded into the pan of the instrument's microbalance. The sample was isothermally maintained at 22 ° C, and the sample mass was monitored throughout the measurement. After 0% RH was purged to remove surface water, the relative humidity (RH) in the sample chamber was ramped up to 90% RH at a constant rate of 4% RH per hour. The sample was kept at 90% RH for one hour, and then RH was reduced to 0% as a step change. The sample was kept at 0% RH for one hour, and the measurement was terminated afterwards.

[0316] Dynamic Scanning Calorimetry (DSC) : Powdered samples were analyzed using dynamic scanning calorimetry. 5 to 10 mg of powdered sample was loaded into an aluminum crucible and hermetically sealed. The crucible was loaded into the instrument and the heat flow into the sample was monitored while the temperature was ramped up from -80 to 200°C, optionally from 20 to 180°C, at a constant rate of 5°C / min.

[0317] USP <790> :According to USP <790> The sample of dissolved particles was visually observed under the standard lighting conditions of greater than 2000 lux on white and black backgrounds. Matte treated high density polyethylene sheet was selected as the background to reduce glare. The illumination at the observation point was confirmed with an illuminometer (Dr. Meter, LX1330B). The sample was rotated and then kept at the background and observed for 5 seconds.

[0318] Flocculation volume Flocculation volume can be measured by suspending the particles in the desired liquid carrier at an appropriate concentration. The suspension is then usually stirred by shaking or mechanical means to ensure that the particles are evenly dispersed in the fluid at the beginning of the measurement. After stirring stops, the timing starts immediately. The mixture is allowed to stand, and the degree of precipitation or settling of the solids over time is observed and recorded.

[0319] Example 1

[0320] General protocol for protein particle preparation: 1. The protein aqueous solution is adjusted to a final concentration of 50-160 mg / mL. 2. Excipients are then added to the resulting solution, and 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) is added to a container (2 mL to 200 L container). 4. The protein feed solution is then added to the container at various flow rates (e.g., up to 100 mL / min). 5. The mixture is then stirred (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 produce aqueous droplets, which are then dehydrated (time depends on the dehydrating solvent volume, rpm and flow rate of the mixture) to form protein particles suspended in the mixture. 6. The protein particles are then separated from the liquid mixture (e.g., filtered, centrifuged and decanted, etc.). 7. The liquid mixture (aqueous liquid and dehydrating solvent) is removed. 8. Remove residual solvent (e.g., aqueous liquid and dehydration solvent) from the protein particles (e.g., vacuum drying, gas drying, gas sparging, extraction solvent, etc.), and then adjust the moisture content in the particles (e.g., wet gas, extraction solvent, etc.). 9. Then dissolve a portion of the protein particles in DI water and incubate to completely redissolve for characterization. 10. Then analyze the solution to measure protein quality (using FLOWCAM TM SvP analysis and soluble aggregates (e.g., SEC), fragmentation (e.g., SEC), changes in charge variants (e.g., ion exchange chromatography ( SCEX )) analysis) and 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 to 93% (w / w)). Particles with up to 85% protein loading showed improved stability compared to the starting feed aqueous solution.

[0321] Preparation of Ab particles within the target size and dispersity range : Rituximab, trastuzumab, human or bovine IgG particles were formed according to the general protocol and 10 mg of particles were provided for analysis. Count-weighted particle size analysis (FLOWCAM TM ) reported the average particle sizes D10, D50 and D90. The average particle sizes D10, D50 and D90 of the particles were determined to be an average particle size of 5-50 μm, and D90 < 60 μm. The particles had the following particle size statistics: Average diameter = 5-50 μm. SEM of each batch of particles was obtained. Figure 1As shown, images were taken at 1000X magnification. Image processing and statistics were performed using ImageJ analysis, count weighting and providing protein particles with desired physical properties (e.g., circularity, internal void space, protein weight, stability and quality, etc.) for suspension studies.

[0322] Determination of aggregation, fragmentation and charge variant changes in particles by SEC : Dry powder samples were dissolved to 5% (w / v) in ultrapure water and shaken at 60RPM. After determining the protein concentration in triplicate, the samples were diluted to 1 mg / mL with PBS and syringe filtered into HPLC vials. The samples in the carrier liquid were resuspended and each sample was immediately centrifuged at 500RPM, and the resulting carrier liquid supernatant was aspirated (and discarded) without disturbing the settled particles. After adding water to the particles and mixing, the samples were shaken at 60RPM. After the initial protein concentration was determined (from the bottom or water layer), each sample was diluted with PBS and the protein concentration was measured in triplicate (from the bottom or water layer). Each sample was then diluted with PBS and syringe filtered into an HPLC vial, and the needle was discarded before filtering (to prevent carrier liquid transfer). All samples were analyzed using AGILENT TM 1260Infinity II bio-inert LC system and TSKgel SuperSW HTP column (4μm, 4.6mmID×150mmL) were analyzed, and the column was balanced with 200mM arginine-HCl, 100mM sodium phosphate, pH 6.5. The autosampler and column chamber were maintained at 4°C and 20°C, respectively, and the UV absorbance at 280nm was monitored. The run time was 10 minutes. The area % of the integrated high molecular weight (aggregates), monomers and low molecular weight peaks were reported.

[0323] Determination of aggregation, fragmentation and charge variant changes in particles by SCEX : Dry powder samples were dissolved to 5% (w / v) in ultrapure water and shaken at 60 RPM. After determining the protein concentration in triplicate, the samples were diluted with ultrapure water and syringe filtered into HPLC vials. The samples in the carrier liquid were resuspended and 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, the samples were shaken at 60 RPM. After the initial protein concentration was determined (from the bottom or water layer), each sample was diluted with ultrapure water and the protein concentration was measured in triplicate (from the bottom or water layer). Each sample was then diluted with ultrapure water and syringe filtered into an HPLC vial, and the needle was discarded before filtering (to prevent carrier liquid transfer). All samples were analyzed using AGILENT TM 1260 Infinity II Bio-inert LC System and THERMO SCIENTIFIC MABPAC TMThe analysis was performed using an SCX-10RS column (5 μm, 2.1 mm ID×150 mm L) with 100% mobile phase A (THERMO SCIENTIFIC TM 1X CX-1 pH gradient buffer A) equilibrated. The gradient was 0-20% mobile phase B (THERMO SCIENTIFIC TM 1X CX-1 pH gradient buffer B). The autosampler and column compartment were maintained at 4°C and 30°C, respectively, and UV absorbance at 280 nm was monitored. The area % of the integrated acidic, neutral (main) and basic peaks are reported.

[0324] Example 2

[0325] General scheme 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) are made into a suspension 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), saturated vegetable fatty acids C8 and C10 propylene glycol diesters (PGD), triacetin, caprylic acid triglyceride) at a concentration of 200-700 mg / mL. This is achieved by adding protein particles to the organic carrier liquid and using a mixer (e.g., rotor stator, etc.) to produce a dilute suspension. The dilute suspension is filtered through a 60 μm filter and the suspension is adjusted to the desired protein concentration. A flocculant is added to this suspension to reach a total flocculant concentration of up to 10 mg / mL, wherein the protein concentration is 200-800 mg / mL. This suspension was initially mixed for 1 minute using a vortex and / or mechanical mixing to produce the desired flocculation volume which remained stable for at least 1 month.

[0326] 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)) are made into a suspension in ethyl oleate at a concentration of 500 mg / mL. This is achieved by adding 19-23 g of particles to about 450 mL of ethyl oleate and using a rotor stator mixer to produce a diluted suspension. The diluted suspension is sieved through a 60 μm filter and adjusted to a HIgG concentration of 500 mg / mL by centrifugation. In the aliquot of this suspension, add flocculant (polysorbate 80) to reach a final total concentration of up to 10mg / mL with a pipette, wherein the HIgG concentration is 500mg / mL. This suspension is vortexed, and then mechanically mixed for 1 minute, to produce a stable suspension (flocculation volume), which remains consistent for at least one month compared to the particle suspension without flocculant. HIgG and BIgG can be used interchangeably. Complete the characterization of protein according to Example 1, and compared with the initial feed aqueous solution, it demonstrates improved stability.

[0327] Rituximab granule composition : Rituximab particles (98% protein: 2% histidine or 80% protein: 2% histidine: 18% arginine) were made into suspensions in ethyl oleate at concentrations of 500 and 700 mg / mL. This was achieved by adding the particles to approximately 450 mL of ethyl oleate and using a rotor stator mixer to produce a dilute suspension. The dilute suspension was sieved through a 60 μm filter and adjusted to a rituximab concentration of 500 or 700 mg / mL by centrifugation. A flocculant (polysorbate 80) was added to aliquots of this suspension with a pipette to achieve a final total concentration of up to 10 mg / mL, with a rituximab concentration of 500 or 700 mg / mL. This suspension was vortexed and then mechanically mixed for 1 minute to produce a stable suspension (flocculated volume) that remained consistent for at least one month compared to a particle suspension without a flocculant. Characterization of the protein was completed according to Example 1 and showed improved stability compared to the starting aqueous feed solution.

[0328] Trastuzumab granule composition:According to the rituximab protocol, trastuzumab particles (71% protein: 2% histidine: 26% arginine: 1% PS80) were made into suspensions in ethyl oleate at a concentration of 500 or 700 mg / mL. According to the rituximab protocol, a stable suspension (flocculation volume) that remained consistent for at least one month was produced. Characterization was completed according to Example 1, and compared to the starting feed aqueous solution, it showed improved stability.

[0329] Example 3

[0330] Protein particle composition with high concentration, stable flocculation volume and low injection force :

[0331] The units "mPa·s" and "cP" are used interchangeably in the broadest sense.

[0332] Rheology : Suspensions of rituximab, trastuzumab, bovine IgG or human IgG particles with the following properties were produced: 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 in carrier liquids of varying concentrations. When the concentration was>300 mg / mL, the viscosity measured was about 20 cP (mPa·s). Even higher concentrations can be achieved when lower viscosity carriers are considered.

[0333] Rheology (Day 0, Day 7, Day 30 at 40°C) : Track the viscosity of the suspension of particles (rituximab, trastuzumab, bovine IgG or human IgG) over time to ensure that it does not change by more than 5% during the 7-day and 30-day accelerated storage period. At each time point, the viscosity of the suspension was <20 cP (mPa·s) with a protein loading >250 mg / mL. Based on the preparation of a 280 mg / mL protein suspension, the concentration condition was met. The day 0 sample was measured immediately. The day 7 and day 30 samples were aged according to the above aging procedure. At 1000 s -1 The measurements were performed at a shear rate of 1.5 Å. On day 0, the viscosity of the suspension was measured to be 16.87 cP. On day 7, the viscosity decreased to 16.33 cP. On day 30, the viscosity increased by 29% compared to day 0 to 21.83 cP, and was slightly above the 20 cP limit. However, with such a small plate (25 mm), small instrument gap (150 microns), and small sample volume, it is estimated that the error in the measurement could be as high as 30%.

[0334] Karl Fischer titration : Karl Fischer titration was used to determine the water content of the solid particles (rituximab, trastuzumab, bovine IgG or human IgG) to define the component composition of the particles. For each of rituximab, trastuzumab, bovine IgG or human IgG, the water content of the particles was measured, recorded and determined to be 1-6 wt%.

[0335] Determination of viscosity : A 25 mm 3 degree cone (CP25-3, Anton Paar) was loaded onto a rheometer (Anton Paar Modular Compact Rheometer (MCR) 92 Series). The plate was warmed to 25°C. The suspension with flocculant was vortexed until visually homogeneous. 250 uL was pipetted onto the substrate and the sample was measured at a shear rate of 950 1 / s.

[0336] Determination of protein content : The particle suspension with flocculant was dispersed by vortexing, diluted to 2.5% (v / v) in ultrapure water, and shaken at 60RPM for 30 minutes; the corresponding dry powder sample was dissolved in ultrapure water to 5% (w / v) and shaken at 60RPM for 30 minutes. After determining the protein concentration in triplicate (from the bottom or water layer of the suspension), the mass percentage of protein in the particles was calculated by dividing the product of the average redissolved protein particle concentration and the redissolved volume by the mass of the microparticles. The theoretical mass of the particles in the suspension was then calculated by dividing the average concentration of the suspension (corrected by the aqueous / formulation carrier liquid dilution factor) by this mass percentage of protein in the particles. The results are comparable to the protein content shown in Examples 1 and 2.

[0337] Dissolve : The dissolution rate of each suspension was recorded at day 0, day 7 and day 30. Dissolution was recorded at different time points, confirming that the particles dissolved in about 20 minutes and the suspension dissolved in about 60-80 minutes. The particles and suspension were dissolved in PBS to a final concentration of 10 mg / mL and shaken on a rocker. The dissolution of the particles at day 0, day 7 and day 30 was also stored at 40°C.

[0338] USP <790> : To determine the presence of visible particles in a sample of dissolved particles. USP <790> Used to determine if particles in the "visible" range (>100 μm) are present. Observation of dissolved particles is used to assess the presence of visible particles. Brief observations (5 seconds) were performed with white and black backgrounds under appropriate lighting. Some potential small particles were observed, but were difficult to see with the naked eye and might be classified as sub-visible particles. Given that these particles might be considered sub-visible, other methods have been used to further investigate, as described below.

[0339] Sub-visible particle analysis: The presence of subvisible particulate matter in dissolved granules was investigated. Subvisible particle analysis was performed after granules were dissolved. This data was compared to other competitive particle formation techniques. SvP analysis was performed by a particle analyzer, where the particle count was adjusted for background signal in control samples. The subvisible particle counts measured in the samples were lower than any other completion technique (including standard lyophilization).

[0340] Flocculation volume : BIgG particles were added to 3 mL of ethyl oleate, followed by 0.1 wt% to 0.01 wt% of flocculant (lecithin or PS 80) to form a protein concentration of 500 mg / mL. The suspension was first mixed and the flocculated volume was calculated and compared with a suspension without flocculant. Figure 2 The effect of flocculants on particle aggregation and flocculation volume is shown. 0.01 wt% and 0.1 wt% lecithin can reduce particle aggregation and flocculation volume. PS 80.01 wt% and 0.1 wt% increase particle aggregation and flocculation volume compared to suspensions without flocculants. In both cases, no reduction in flocculation volume was observed and it remained stable at 25C for at least three months.

[0341] Injectability :Measure the force of injecting flocculant into suspension by needle, and verify that injection force remains the same after 24 hours, without resuspension before injection. As obtained above, the suspension of BIgG particles with a concentration of 500mg / mL of protein and 0.1 wt% PS80 in EO is loaded into a 5mL syringe with 18-G needle (3 inches). The injection force is less than 2.0N and remains the same after storage for 24 hours. As a reference, the actuation force of an empty syringe equipped with a syringe needle is compared using a 15mPa·s standard, and the results show that the injection force is less than 2.0N. These studies show that the force of the injection composition remains stable for at least one month.

[0342] Example 4

[0343] Particle and suspension quality studies :

[0344] design : A particle suspension with a flocculant was obtained using a procedure similar to that of Examples 1-3. The stability of the protein particle suspension with a flocculant was evaluated for three months 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., a starting feed aqueous solution).

[0345] Composition: A particle suspension with flocculant was obtained using a procedure similar to Examples 1-3, wherein the flocculant (600 mg / mL protein and 0.1 mg / mL PS80; 74% protein: 2% histidine: 24% arginine: 1% PS80) was formulated into particles that were then suspended in ethyl oleate.

[0346] Buffer exchange by tangential flow filtration (TFF) Protein feed solutions were prepared by diafiltration into the desired buffer mixtures. Feed preparation was performed using a KrosFlow KR2i TFF system (Repligen) equipped with a hollow fiber filtration module (MiniKros sampler). Multiple diafiltration volume exchanges were performed for each formulation with the appropriate buffer.

[0347] FIB-SEM imaging :Using scanning electron microscope (HITACHI TM The particles were imaged using a 1000nm RT-PCR (TM-1000). Samples of the particles were mounted on a bonding stage for analysis. Images were captured at different magnifications using an accelerating voltage of 15 kV.

[0348] Buffer exchange by tangential flow filtration (TFF) Protein feed solutions were prepared by diafiltration into the desired buffer mixtures. Feed preparation was performed using a KrosFlow KR2i TFF system (Riprich) equipped with a hollow fiber filtration module (MiniKros Sampler). Multiple diafiltration volume exchanges were performed for each formulation with the appropriate buffer.

[0349] SEM imaging :Using scanning electron microscope (HITACHI TM The particles were imaged using a 1000nm RT-PCR (TM-1000). Samples of the particles were mounted on a bonding stage for analysis. Images were captured at different magnifications using an accelerating voltage of 15 kV.

[0350] Karl Fischer Coulometric Method :Use with 860KF THERMOPREP TM The moisture content was tested by Karl Fischer analysis using a MetroOhm oven (899 coulometer). The particles were heated to 165°C in an oven and the released water was determined by coulometric method.

[0351] Particle dissolution : Add water to the dried particle sample to produce the final protein concentration. Place the sample on a nutating mixer at 60 RPM for a certain period of time. Record the final dissolved concentration by taking an aliquot from the sample and measuring the absorbance at 280 nm (using the extinction coefficient E1% = 1.69 L·g-1·cm-1).

[0352] Turbidity: Transfer an aliquot of the reconstituted particle solution into a 1-cm path length cuvette. Using the NANODROP TM One UV-VIS Spectrophotometer (THERMO SCIENTIFIC TM ) The absorbance at 405 nm was recorded. The results showed that the turbidity was improved compared to the starting feed aqueous solution and the particles.

[0353] Size Exclusion Chromatography (SEC) Measurements : On Tosoh TSKgel SuperSW mAb HTP (4.6mm ID×15cmL) column, use the mobile phase (pH6.5) comprising 100mM sodium dihydrogen phosphate and 200mM L-arginine monohydrochloride, with the flow rate of about 0.35 ml / min to run the injection of sample for 10 minutes. Manually check the peak to ensure accurate identification, and analyze by automatic integration using parameters known in the art.

[0354] Strong Cation Exchange Chromatography (SCEX) : The solution was injected into the MILLI-Q TM For samples in water, the gradient method started with 100% mobile phase pH gradient buffer A to 100% mobile phase pH gradient buffer B, followed by a MAbPac TM The total run time was 40 minutes, washed and re-equilibrated on a SCX-10RS analytical column (2.1 mm ID x 15 cm L, 5 μm column). Peaks were manually checked to ensure accurate identification and analyzed by automated integration using parameters known in the art.

[0355] Hydrophobic Interaction Chromatography (HIC) Injections of samples dissolved in a diluent containing 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 using a gradient starting from 50% mobile phase A containing 2 M ammonium sulfate, 50 mM sodium dihydrogen phosphate dihydrate, pH 6.0 to 100% mobile phase B containing 50 mM sodium dihydrogen phosphate dihydrate, pH 6.0, followed by washing and re-equilibration with 50% mobile phase A in MAbPac TM The total run time on a HIC-20 HPLC column (5 μm, 4.6 mm ID x 25 cm L column) was 60 minutes. Peaks were manually checked to ensure accurate identification and analyzed by automated integration using parameters known in the art.

[0356] Particle size screening (laser diffraction):Particle size analysis was performed by laser diffraction using Horiba LA-960S. The dry particles were suspended in isopropanol at a concentration of about 0.1 mg / mL. The particle suspension was ultrasonically treated in the particle measuring instrument to ensure uniformity, and then circulated and stirred by the Horiba particle size analyzer. The mobile phase of isopropanol was used to perform particle size analysis, and the volume average particle size distribution was calculated.

[0357] Microfluidic Imaging :Flow imaging microscopy (FLOWCAM TM , Fluid Imaging Technologies) to quantify subvisible particles in protein LDS and particle formulations. Particles were first redissolved using the particle solubilization method described above and diluted to 1 mg / mL with ultrapure water. For analysis, aqueous samples were introduced at a flow rate of 0.15 mL / min. Particle counts per mg of protein were recorded and reported.

[0358] result :The stability of dry particles and particle suspensions with flocculants was evaluated together with protein liquid drug substances (LDS). Stability was tracked at 5, 25 and 40°C, and data was collected at 7 days, 14 days, 1 month, 3 months, 6 months, 9 months and 12 months. Particle stability, protein stability of LDS and suspensions were measured at each time and temperature. Analysis of particles and particle suspensions confirmed that protein quality remained constant as measured by monomer curve (SEC), maintenance of charge variant curve (CEX), isoform / hypothetical oxidation (HIC) and colloidal stability (turbidity and sub-visible particles). In each case, protein feed solutions were measured for particles and particle suspensions with flocculants. Particles stored in the form of dry powders and particle suspensions at 5, 25 and 40°C showed smooth and spherical morphology. No changes in particle morphology or particle size distribution were observed over the course of three months at these temperatures. The moisture content of the particles remained constant at all storage temperatures and time lengths. Analysis of protein aggregates by SEC was measured for up to 12 months at 5, 25 and 40°C. A comparison of the effect of excipient selection (histidine, arginine.HCl and PS80) on the stability of hIgG particles (without flocculant) was performed relative to Δ(Mon+Dim)% and Δ(Dim+Agg)% for standards and stored at 40°C for 12 months to assess aggregation over time compared to LDS samples. Figure 3 As shown, the LDS samples stored at 40°C for 12 months exhibited high protein aggregation compared to the hIgG particles and suspension samples with flocculants stored at the same temperature (40°C for 12 months).

[0359] The low aggregation rate of the particle suspension with flocculant proves that its stability is improved compared with protein LDS. At 5, 25 and 40 ° C, the charge variation curve of the protein was measured by SCEX for up to 90 days. No obvious changes were observed for the particles and the particle suspension with flocculant, which proves that the stability at 40 ° C is improved compared with the protein LDS. HIC is used to measure the potential oxidation of proteins. At 5, 25 and 40 ° C, the assumed global oxidation degree of proteins was measured by HIC for up to 90 days. No obvious changes were observed, indicating that the particles and the particle suspension with flocculant showed improved stability to oxidative stress at 25 and 40 ° C compared with the protein LDS at the same temperature and time. In order to further explore the protein quality of the particles redissolved in aqueous media, visible and sub-visible particles were analyzed after storage at different temperatures. After dissolution, the protein solution was basically free of visible particles and sub-visible particles after storage at 40 ° C for up to 90 days. For the particle suspension with flocculant, there was no significant change in protein concentration, viscosity and injection force at all storage times and temperatures.

[0360] Example 5

[0361] Stability of proteins with flocculants determined by structure (day 0, day 7, day 30 at 40°C) :

[0362] Size Exclusion Chromatography (SEC) : The aim was to collect SEC data to evaluate the degradation of protein monomers after 30 days of storage at 40°C in the presence of a flocculant. SEC was performed on protein liquid drug substance (LDS), particles and suspensions with a flocculant. Monomer, aggregate and fragment data were obtained for each sample. SEC data were recorded on day 0, day 7 and day 30 after storage at 40°C and good stability of the protein monomer was demonstrated by treatment and storage with a flocculant. The 30 day sample of LDS showed poorer preservation of the monomer compared to the particle suspension with a flocculant, with each of the suspensions having a monomer preservation of >98%.

[0363] Cation Exchange Chromatography (CIEX)CIEX data were collected to evaluate the distribution of charge variant species and their changes over storage for 30 days at 40°C in the presence of a flocculant. CIEX was performed on LDS, particles, and suspensions with a flocculant. Acidic, major, and basic species data were obtained and analyzed for each sample. The CIEX data points were selected to highlight the acidic shift experienced by the labeled formulations compared to the absence of such changes in the suspension after 30 days of aging. Proteins from the suspension were similar to untreated material, while LDS experienced high degradation. CIEX data were recorded as recommended and good stability of the charge variants was demonstrated by storage with a flocculant on days 0, 7, and 30 after storage at 40°C. Due to the acidic shift in the variants, the 30-day sample of LDS had extremely low preservation of the charge variant distribution compared to the particle suspension with a flocculant.

[0364] Aggregation, fragmentation, and charge variants of proteins : Unless otherwise stated, aggregation and fragmentation data were collected using size exclusion chromatography (HPLC-SEC) according to Examples 1-3. Unless otherwise stated, distribution of charge variant species and their changes were collected using strong cation exchange chromatography (CIEX) according to Examples 1-3. The results show that the stability of the suspension with flocculant is improved compared to the starting LDS.

[0365] Example 6

[0366] Flocculant and protein concentration studies :

[0367] The average gliding force was plotted as a function of particle suspension concentration (74% protein: 2% histidine: 24% arginine: 1% PS80) up to 700 mg / mL. 20 N (Newtons) was accepted as a reference for the typical gliding force limit.

[0368] Flocculation volume by increasing flocculant and protein concentration : Protein particle suspensions were generated using ethyl oleate as the liquid carrier, using BIgG at concentrations ranging from 300 to 700 mg / mL, with flocculant (PS80) concentrations ranging from 0 to 10 mg / mL, and stored for 24 hours. Figure 4 A list of flocculant and protein concentration parameters for the flocculation volume study is shown. Table 1 shows that with increasing concentrations of BIgG and PS80, the flocculation volume increases.

[0369] Table 1

[0370]

[0371] Syringability by increasing flocculant and protein concentrationProtein particle suspensions using BIgG were generated at concentrations ranging from 500 to 700 mg / mL using ethyl oleate as the liquid carrier, with flocculant (PS80) concentrations ranging from 0 to 10 mg / mL, using 20-gauge and 22-gauge 13 mm (half-inch) needles (Japan BioProducts) attached to 1 mL syringes. Table 2 shows that the flocculation volume increased with increasing concentrations of BIgG and PS80, with no substantial change in injection force.

[0372] Table 2

[0373]

[0374] Use of injectable caprylic triglyceride : A protein particle suspension using BIgG was produced at a concentration of 620 mg / mL (74% protein: 2% histidine: 24% arginine: 1% PS80) using caprylic triglyceride as a carrier liquid, wherein the concentration of the flocculant (PS80) was 0.1 mg / mL. After 24 hours, no change in the flocculation volume was observed. Using a 22-gauge 13 mm (half-inch) needle (Japan Biopharmaceuticals Co., Ltd.) attached to a 1 mL syringe, the injection force was 5.2 N and the speed was 0.1 mL / sec, as Figure 5 shown.

[0375] Flocculation volume and storage : Protein particle suspensions using BIgG were produced at a concentration of 556 mg / mL (74% protein: 2% histidine: 24% arginine: 1% PS80) using ethyl oleate as the liquid carrier, with flocculant (PS80) concentrations of 0 mg / mL and 10 mg / mL, and stored at 4°C to 25°C for 1 month. Figure 6 As shown, no change in flocculation volume was observed at 4°C or 25°C after PS80 was added to the suspension 1 month later. In addition, the injection force remained essentially the same (up to 14 seconds) at 2.4 N using a 20-gauge 13 mm (half-inch) needle (Nippon Biosciences, Inc.) attached to a 1 mL syringe. Figure 7 .

[0376] Injection force by needle size: Protein particle suspensions using BIgG were produced 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 (Nippon Biologics, Inc.) attached to 1 mL syringes. After 24 hours, the flocculated volume remained at 100% with no substantial change in injection force for all three needle sizes (3 N for a 20-gauge needle, 6 N for a 25-gauge needle, and 16 N for a 27-gauge needle). See Figure 8 .

[0377] Injection force using 27-gauge UTW needle : Protein particle suspension using BIgG was produced 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 Biopharmaceuticals, Inc.) attached to a 1 mL syringe. After 24 hours, the flocculated volume remained at 100%, with no substantial change in the injection force (17.4 N). Fig. 9 Shown is the injection force versus injection time using a 27 gauge UTW needle at a protein concentration of 625 mg / mL.

[0378] Capsule storage : A protein particle suspension using BIgG was prepared at a concentration of 556 mg / mL using ethyl oleate as a liquid carrier, with a flocculant (PS80) concentration of 10 mg / mL, and added to two gelatin capsules (18 mm long, Medlab) without bubbles. After 1 month, the flocculation volume remained at 100%, with no observable sedimentation of the protein particles. See Fig. 10A and 10B .

[0379] Example 7

[0380] Pharmacokinetics (PK): Subcutaneous administration of Ab microparticle suspension showed higher bioavailability than aqueous Ab injection Spend :

[0381] Composition: The scope of the study involved four cohorts with the following compositions: 1) IV Ab aqueous rituximab: 30 mg / mL, 2) SC Ab aqueous solution: 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 as a particle composition and then suspended in ethyl oleate (non-aqueous carrier vehicle) with a flocculant (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).

[0382] animal Female wild-type albino Sprague-Dawley rats (Albino Sprague Dawley Rats, Charles River Lab) were housed in the animal facility at Tufts University Comparative Medicine Services (Tufts CMS). All uses of animals were carried out according to the "Guide for the care and use of laboratory animals" of the National Research Council (National Research Council), and were carried out according to the detailed written scheme approved by the Institutional Animal Care and Use Committee (IACUC).

[0383] Dosing, Study Design, and Analysis: All particle composition injections were done using a 100 μL Hamilton glass syringe with a 27-gauge needle. No protocol for resuspending the particles in the syringe was implemented. All aqueous and non-aqueous vehicle injections were done using a 300 μL insulin syringe with a staked-in 30-gauge needle. No protocol for resuspending the particles in the syringe was implemented. Sprague-Dawley rats were used for this study. The following study design was used: 1) IV Ab aqueous solution: dose = 10 mg / kg; volume = 100 μL; n = 5, 2) SC Ab aqueous solution: 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 0 hours, 3 hours, 6 hours, 9 hours, 1 day, 2 days, 3 days, 5 days, 6 days, 9 days, 12 days (14 days, 16 days, 19 days, 21 days and 23 days: these time points were below the detection limit of the assay utilized). At all data points, blood was collected from each rat using EDTA capillaries (purple) from the tail vein bleed. Plasma obtained from the blood was diluted to 10,000 times for analysis using ELISA. Plasma samples were provided by a third-party contract research organization (Ray Biotech: The test article rituximab SC injection: AUC 5.8 (hours*mg / mL), Cmax 34.0 (μg / mL), Tmax 57.6 (hours). The test article rituximab microparticle suspension SC injection in ethyl oleate (EO): AUC 6.3 (hours*mg / mL), Cmax 36.7 (μg / mL), Tmax 67.2 (hours). Fig.11 As shown, rituximab microparticle suspension showed higher bioavailability than aqueous rituximab injection.

[0384] Toxicity and local tolerance studies and analysis : The injection site was evaluated for edema (swelling) and erythema (redness) before and up to 3 days after injection. The site was scored based on the severity of the response using Draize scoring (0-4 maximum). No abnormal findings were observed in any of the 5 animals in the composition group (100 mg / kg, 500 mg / mL) on Day 0 (before injection), Day 1 (after injection), and Day 7 (after injection).

[0385] Erythema and eschar formation: no erythema – 0; very mild erythema (barely noticeable) – 1; well-defined erythema – 2; moderate to severe erythema – 3; severe erythema (beefy redness) to eschar formation, prevention of erythema – 4.

[0386] Edema formation: no edema – 0; very mild edema (barely noticeable) – 1; mild edema (clearly defined edge of area by clear elevation) – 2; moderate edema (elevation approximately 1 mm) – 3; severe edema (elevation greater than 1 mm and extending beyond the exposed area) – 4.

[0387] Dresser score: The presence or absence of findings were recorded for individual animals on Day 0 (before injection), Day 1 (after injection), and Day 7 (after injection). All 5 animals in the group had zero edema and erythema scores within 3 days after injection, indicating that the composition samples were well tolerated and non-toxic.

[0388] Detailed clinical observation : The animals were removed from the cages and observed. These included, but were not limited to, evaluation of the skin, fur, eyes, ears, nose, oral cavity, chest, abdomen, external genitalia, limbs and feet, respiratory and circulatory effects, autonomic effects (such as salivation), nervous system effects (including tremors, convulsions, responsiveness to treatment, abnormal behavior). No abnormal findings were observed in any of the 5 animals in the composition groups (100 mg / kg, 500 mg / mL) on Day 0 (before injection), Day 1 (after injection), and Day 7 (after injection), indicating that the composition samples were well tolerated with no observable toxicity.

[0389] Example 8

[0390] Immunogenicity: Subcutaneous administration of Ab microparticle suspension showed no immune response :

[0391] Composition The study involved four cohorts with the following compositions: 1) SC vehicle liquid control: ethyl oleate; 10 μL, 2) SC mAb aqueous solution: 20 mg / mL rituximab biosimilar (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; 10 μL, 3) SC mAb microparticle suspension: 20 mg / mL rituximab biosimilar (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 were formulated into particles and then suspended in ethyl oleate (non-aqueous carrier vehicle) with a flocculant (PS80) at a concentration of 10 mg / mL; 10 μL and 4) SC degraded aqueous mAb: rituximab biosimilar (anti-CD-20 antibody), subjected to heating and stirring to produce a positive control sample; 10 μL.

[0392] animal Female wild-type Balb / c mice (Charles River Laboratories) 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) of Tufts CMS.

[0393] Dosing, Study Design, and Analysis : All particle composition injections were performed using a 100 μL Hamilton glass syringe with a 27-gauge needle. All aqueous and non-aqueous vehicle injections were performed using a 300 μL insulin syringe with a spiked 30-gauge needle. No protocol for resuspending particles in the syringe was implemented. Balb / c female mice (Charles River Laboratories), 10 weeks old, were used for this study. The following study design was used: 1) SC vehicle liquid control: volume = 10 μL; n = 10, 2) SC rituximab aqueous solution: 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 degraded rituximab aqueous solution: dose = 10 mg / kg, volume = 10 μL; n = 10. Plasma samples were collected on days 0 and 35. At all data points, blood was collected from each mouse using an EDTA capillary from the facial vein bleed. Plasma obtained from blood was diluted 2,500 times for use with the Meso Scale Analysis of electrochemiluminescence (MSD-ECL) platform. The master mixture was made by diluting SULFO-TAG and biotinylated rituximab with equal concentrations (0.5 μg / ml) in an assay diluent (1% MSD blocker A in PBS with 0.02% Tween 20). The ADA standard curve was made 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 mixture was added to each well of a round-bottom 96-well polypropylene plate. Then, 25 μL of ADA standards or 4% plasma samples were added to each well. The plate was 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 plate was sealed and incubated with moderate shaking at room temperature for at least 30 minutes, then washed with 200 μL PBST. 50 μL of solution was transferred from each well of the polypropylene plate to the streptavidin plate. The plate was then sealed and incubated with shaking at 400-700 rpm for 1 hour at room temperature, followed by washing 3 times with 200 μL PBST. 150 μL of 2X read buffer was added to each well. The plate was analyzed using an MSD QUICKPLEX instrument. The results of this study indicate that subcutaneous administration of Ab microparticle suspensions showed no immune response based on repeated doses of anti-drug antibodies (immunogenicity) to rituximab.

[0394] Example 9

[0395] Administration of hyaluronidase allows for administration of the volume and volume of the composition with the flocculant by injection via a hypodermic syringe. Increased fluid dispersion :

[0396] Particle and suspension production was performed using procedures similar to those described in Examples 1-4. Particle formulation: HIgG74% protein: 2% histidine: 24% arginine: 1% PS80 provided smooth and spherical particles with a particle water content of 3%. FIB-SEM showed that the particles had no internal void space. Suspension formulation: carrier liquid ethyl oleate, protein content 100 mg / mL, 1% PS80. The flocculation volume remained stable at 25°C for at least 1 month. SVP>2μm, measured by Aura normalized per mg protein (1.8E5), SEC 98.9% monomer data indicated that the particles remained stable at about 25°C for at least 1 month.

[0397] Composition: The scope of the study involved one pork belly sample 8" x 18" double bagged (Savenor's, Boston) and equilibrated to physiological temperature with two sets of injection sites using the following compositions: Group 1 injection sites of PBS solution; and Group 2 injection sites of SC Ab suspension administered after hyaluronidase pre-injection: i) 2000U of 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 of protein. No protocol for resuspending the particles in the syringe was implemented.

[0398] Dosing, Study Design, and Analysis : Allow a five-pound portion of skinned pork belly to reach physiological temperature. Use alcohol to thoroughly clean the skin side and then wipe dry with sterile gauze. No scheme for resuspending particles in a syringe was implemented. While the skin was taut, a PBS solution was injected subcutaneously under the skin at a 45-degree angle using a 24mL Henke-Ject syringe and a 27G UTW needle (Group 1). The suspension was distributed until sufficient back pressure to prevent further distribution of the suspension was generated. The volume distributed was 1.4mL, and the average injection rate was 0.025 ml / sec. The experiment was repeated (Group 2) by subcutaneous administration of SC Ab suspension after hyaluronidase pre-injection, using a 1mL tuberculin syringe and a 25G UTW needle to pre-inject 1mL of hyaluronidase mixture subcutaneously under the skin at a 45-degree angle. After a period of about 5-10 minutes, SC Ab suspension was subcutaneously injected into the same injection site at a 45-degree angle using a 24mL Henke-Ject syringe and a 25G UTW needle. The suspension was dispensed until sufficient back pressure prevented further dispensing of the suspension. The average volume was 5.3 mL, and the average injection rate was 0.045 mL / sec. The endpoints of the study included injection volume measurement and qualitative assessment of the injection site. All injections were performed using the same injection force.

[0399] result: The purpose of this study was to determine the maximum feasible volume that could be administered when given subcutaneously as a single dose. The experiments indicated that the injection volume of SC Ab suspensions administered subcutaneously (total volume 1.4 mL) could be substantially increased by pre-injection of hyaluronidase (total volume 5.3 mL), and that these hyaluronidase injected sites showed an improvement in the injection rate of the Ab particle composition, as determined by the appearance of skin blisters formed at the treatment site, as well as a reduction in injection force (increase in injection rate) and a greater injection volume.

[0400] Example 10

[0401] Particle and suspension production was performed using a similar procedure 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.

[0402] Particle generation and droplet formation (Kinematica / 20mm) studies: Diffuse phase:continuous phase (nBA) ratio 1 / 200, at least 16mL:3.2L, with mixer speed (10000rpm) / 60 seconds mixing. 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 a particle water content of 3.4% ( Fig.12 ). FIB-SEM shows that the particles have no internal void space ( Fig.13 ).

[0403] Ethyl oleate was used as the carrier liquid and the filling volume was 2.56 mL. Suspension dispersion process and parameters: RSM 5000 rpm was used for 120 seconds and filtration (60 um). 10 uL of a reserve 100 mg / mL PS80 solution in ethyl oleate (1% PS80) was added to the suspension and vortexed to mix. At 25 ° C, the flocculation volume remained stable for at least 3 months. 1 mL of the suspension was transferred to a 1 mL Gerresheimer 13 mm syringe and a 25-gauge UTW needle was added to the syringe. The syringe force was measured 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).

[0404] Syringe Force Results . Fig.14The gliding force was shown to be 16 N when a 620 mg / mL rituximab suspension was injected at 0.05 mL / sec.

[0405] Auto-Injector Force Results The syringe was loaded with 620 mg / mL suspension and placed in a test fixture to simulate a spring-driven automatic injector. The spring was capable of ejecting the suspension from the syringe at 16 N.

[0406] Example 11

[0407] Particle and suspension production was performed using a similar procedure 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.

[0408] Particle generation and droplet formation (Kinematica / 20mm) studies: Diffuse phase:continuous phase (nBA) ratio 1 / 200, at least 20mL:4L, with mixer speed (8000rpm) / 60 seconds mixing. Solid-liquid separation by centrifugation and subsequent drying (air 10-75RH) provided smooth and spherical particles with a particle size distribution (D10 / D50 / D90) of 6.6 / 11.4 / 19.3μm and a particle water content of 3% ( Fig.15 ). FIB-SEM shows that the particles have no internal void space ( Fig.16 ). SVP>2μm, measured per mg protein (3.5E5) normalized by Aura, Pro-A, 16.00% oxidation, SCEX (acidic / neutral / basic %) 7.7 / 82.7 / 9.6, SEC 96.6% monomeric data indicated that the particles remained stable for at least 3 months.

[0409] Ethyl oleate was used as the carrier liquid with a fill volume of 3.2 mL. Suspension dispersion process and parameters: Use RSM6000rpm for 15 minutes and filter (60um). 10uL of a stock 100mg / mL PS80 solution in ethyl oleate (1% PS80) was added to the suspension and vortexed to mix. The flocculation volume remained stable for at least 3 months at 25°C. 1 mL of the suspension was transferred to a 2.25 mL Neopak syringe and a 27-gauge STW x 1 / 2" needle was added to the syringe. The syringe force was measured at 0.1 ml / sec (189.4 mm / min).

[0410] Syringe Force Results . Fig.17The gliding force was shown to be 30 N when a 507 mg / mL rituximab suspension was injected at 0.1 mL / sec.

[0411] Example 12

[0412] Trastuzumab particles and suspension production were performed using procedures similar to those in Examples 10 and 11. 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.

[0413] Particle generation and droplet formation (Kinematica / 20mm) studies: Diffuse phase: continuous phase (nBA) ratio 1 / 200, at least 18mL:3.6L, with mixer speed (4000rpm) / 60 seconds mixing. Solid-liquid separation process by centrifugation and subsequent drying (air 10-75RH) provided smooth and spherical particles, SEC monomer data showed that the particles remained stable for at least 3 months.

[0414] Flocculation volume and storage results After 1 month, the flocculation volume remained stable at 100%, with no observable settling of protein particles.

[0415] Syringe Force Results After 1 month, the results showed that the gliding force of the 507 mg / mL trastuzumab suspension did not change.

[0416] Example 13

[0417] Particle and suspension production was performed using a similar procedure as in Examples 1-4. Particle formulation: HIgG 74% protein: 2% histidine: 24% arginine: 1% PS80 provided smooth and spherical particles with a particle water content of 3%. FIB-SEM showed that the particles had no internal void space. Suspension formulation: carrier liquid ethyl oleate, protein content 500 mg / mL, 1% PS80. The flocculation volume remained stable at 25°C for at least 1 month. SVP>2μm, measured by Aura normalized per mg protein (1.8E5), SEC 98.9% monomer data indicated that the particles remained stable at about 25°C for at least 1 month.

[0418] Flocculation volume and storage stability results. After 1 month at 4°C, 25°C and 40°C, the flocculation volume remained stable at 100%, with no observable sedimentation of protein particles. The storage stability of 500 mg / mL HIgG suspensions was tracked at 4°C, 25°C and 40°C, and data was collected at 1 month, 2 months and 3 months, with PS80 concentrations in ethyl oleate of 0 mg / mL, 0.1 mg / mL and 1.0 mg / mL. The data showed that after storage at 4°C, 25°C and 40°C for up to 3 months, the SVP>2μm counts decreased with increasing concentrations of PS80 in ethyl oleate. For 500 mg / mL HIgG suspensions stored at 25°C and 40°C for at least 3 months, the SVP counts were reduced by at least 4-fold by 1.0 mg / mL PS80 in EO.

[0419] Syringe Force Results 1 mL of the suspension was transferred to a 1 mL Schott SyriQ BioPure syringe and a 27 gauge 1 / 2" needle was added to the syringe. The syringe force was measured at 0.1 mL / sec (189.4 mm / min). The syringe force results showed that the gliding force was 16 N when injecting 500 mg / mL HIgG suspension at 0.1 mL / sec.

[0420] Yield stress was measured by parallel plate rheometer (ANTON PAAR TM The results showed that a yield stress was observed, indicating that a stable flocculation volume was formed after the addition of the flocculant, allowing the particle suspension to flow.

[0421] Example 14

[0422] Particle compositions having more than one protein :

[0423] Rituximab and trastuzumab suspension production was performed using a similar procedure as in Examples 10-12. 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) was added to the suspension and vortexed, 221.2 mg of rituximab and 310.5 mg of trastuzumab for a total of 531.7 mg (protein content 400 mg / mL), particle protein percentage 70%.

[0424] Flocculation volume and suspension stability resultsAfter 1 month, the flocculation volume remained stable at 100% with no observable sedimentation of the rituximab and trastuzumab particles. After 1 month, analysis of the particles and particle suspensions confirmed a slight decrease in protein quality as measured by SEC via monomer curves when PS80 was formulated in ethyl oleate and stored at 25 °C for at least 1 month.

[0425] Example 15

[0426] Protein particle composition having at least two carrier liquids :

[0427] Particle and suspension generation was performed using a similar procedure 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, solid loading 452 mg / mL, flocculant: 0 or 2 mg / mL.

[0428] Particle Generation and Droplet Formation (Kinematica / 20mm) Studies: Diffuse phase:continuous phase (nBA) ratio 1 / 200, at least 20mL:4L, with mixer speed (8000rpm) / 60 seconds mixing. 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 a particle water content of 3%. FIB-SEM showed that the particles had no internal void space. SVP>2μm, by Aura

[0429] Normalized per mg protein (3.5E5) measurements, Pro-A, 16.00% oxidation, SCEX (acidic / neutral / basic %) 7.7 / 82.7 / 9.6, SEC 96.6% monomer data indicated that the particles remained stable for at least 3 months.

[0430] 40 / 60 ethyl oleate: sesame oil was used as the carrier liquid with a fill volume of 3.2 mL. Suspension dispersion process and parameters: RSM 6000 rpm for 15 minutes and filtered (60 um). 10 uL of a stock 100 mg / mL PS80 solution in ethyl oleate (1% PS80) was added to the suspension and vortexed to mix. The flocculation volume remained stable for at least 1 month at 25°C (74.8%) compared to the suspension without flocculant (62.3% after 1 month). 1 mL of the suspension was transferred to a 2.25 mL Gerresheimer syringe and a 27 gauge TW x 1 / 2" needle was added to the syringe. The syringe force was measured at 0.1 mL / sec (189.4 mm / min). Store at room temperature, flipped up until no change in flocculation volume was observed (at least 1 month of storage).

[0431] Flocculation volume and storage stability results . The flocculated volume remained stable at 74.8% after 1 month at 25°C, with no observable settling of protein particles, compared to the suspension without flocculant (62.3% after 1 month). The storage stability of a 400 mg / mL HIgG suspension was followed at 25°C, with data collected at 1 month, with PS80 concentrations of 0 mg / mL and 0.1 mg / mL in 40 / 60 ethyl oleate:sesame oil. The data indicate that SVP>2μm counts decreased with increasing concentrations of PS80 formulated in 40 / 60 ethyl oleate:sesame oil after storage at 25°C for up to at least 1 month.

[0432] Syringe Force Results . 1 mL of the suspension was transferred to a 1 mL Gerresheimer syringe and a 27 gauge TW x 1 / 2" needle was added to the syringe. The syringe force was measured at 0.1 mL / sec (189.4 mm / min). The syringe force results showed that the sliding force of the 400 mg / mL HIgG suspension with flocculant using 40 / 60 ethyl oleate:sesame oil as the carrier liquid was 20.1 N when injected at 0.1 mL / sec, and 27.4 N when compared to the suspension without flocculant. The viscosity was found to be 23.3 cP compared to the suspension without flocculant (31.8 cP).

[0433] Yield stress was measured by parallel plate rheometer (ANTON PAAR TM The results showed that a yield stress was observed, indicating that a stable flocculation volume was formed after the addition of the flocculant, allowing the particle suspension to flow.

[0434] Example 16

[0435] Protein granule composition with MCT as carrier liquid :

[0436] Particle and suspension production was performed using a similar procedure 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 MCT, protein content 400 mg / mL, solid loading 452 mg / mL, flocculant: 0 or 2 mg / mL.

[0437] Particle Generation and Droplet Formation (Kinematica / 20mm) Studies: Diffuse phase:continuous phase (nBA) ratio 1 / 200, at least 20mL:4L, with mixer speed (8000rpm) / 60 seconds mixing. 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 a particle water content of 3%. FIB-SEM showed that the particles had no internal void space. SVP>2μm, by Aura

[0438] Normalized per mg protein (3.5E5) measurements, Pro-A, 16.00% oxidation, SCEX (acidic / neutral / basic %) 7.7 / 82.7 / 9.6, SEC 96.6% monomer data indicated that the particles remained stable for at least 3 months.

[0439] MCT was used as the carrier liquid with a fill volume of 3.2 mL. Suspension dispersion process and parameters: RSM 6000 rpm was used for 15 minutes and filtered (60 um). 10 uL of a stock 100 mg / mL PS80 solution in MCT (1% PS80) was added to the suspension and vortexed to mix. The flocculation volume remained stable for at least 1 month at 25°C (87.6%) compared to a suspension without flocculant (65.1% after 1 month). 1 mL of the suspension was transferred to a 2.25 mL Gerresheimer syringe and a 27-gauge TW x 1 / 2" needle was added to the syringe. The syringe force was measured at 0.1 mL / sec (189.4 mm / min). Store at room temperature and flip up until no change in flocculation volume is observed (at least 1 month of storage).

[0440] Flocculation volume and storage stability results. The flocculated volume remained stable at 87.6% at 25°C for 1 month, with no observable settling of protein particles, compared to the suspension without flocculant (65.1% after 1 month). The storage stability of a 400 mg / mL HIgG suspension was followed at 25°C, with data collected at 1 month, with PS80 concentrations of 0 mg / mL and 0.1 mg / mL in MCT. The data indicate that SVP>2 μm counts decreased with increasing concentrations of PS80 formulated in MCT after storage at 25°C for up to at least 1 month.

[0441] Syringe Force Results . 1 mL of the suspension was transferred to a 1 mL Gerresheimer syringe and a 27 gauge TW x 1 / 2" needle was added to the syringe. The syringe force was measured at 0.1 mL / sec (189.4 mm / min). The syringe force results showed that the sliding force of the 400 mg / mL HIgG suspension with flocculant using 40 MCT as the carrier liquid was 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).

[0442] Yield stress was measured by parallel plate rheometer (ANTON PAAR TM The results showed that a yield stress was observed, indicating that a stable flocculation volume was formed after the addition of the flocculant, allowing the particle suspension to flow.

[0443] Incorporated by Reference

[0444] All publications and patents mentioned herein are incorporated by reference in their entirety to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In the event of a conflict, the present application, including any definitions herein, will control.

[0445] Equivalent

[0446] Although specific aspects and embodiments of the present disclosure have been discussed, the above description is illustrative rather than restrictive. Many variations of the present disclosure will become apparent to those skilled in the art after reading this specification and the claims below. The full scope of the present disclosure should be determined by reference to the claims together with the full scope of their equivalents and the specification together with such variations.

Claims

1. A pharmaceutically effective composition comprising: a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein substantially all of the particles comprise at least one therapeutic biologic or a salt thereof; and A flocculant, wherein the concentration of the flocculant in the composition is less than about 50 mg / mL; and wherein the concentration of the therapeutic biologic or salt thereof in the composition is greater than about 250 mg / mL.

2. The composition of claim 1, wherein substantially all of the particles comprise at least one therapeutic biologic.

3. A composition according to claim 1 or 2, wherein the flocculant is ionic.

4. The composition of claim 3, wherein the ionic flocculant is magnesium stearate, sodium lauryl sulfate, sodium stearate, cetyltrimethylammonium bromide, lecithin, or a combination thereof.

5. The composition of claim 1 or 2, wherein the flocculant is nonionic.

6. The composition of claim 5, wherein the nonionic flocculant is polysorbate, alkylphenol ethoxylate, glycerol, polyoxyethylene castor oil, docusate, decyl glucoside, nonoxynol-9, sorbitan esters, sorbitan monooleate, ethanolamine, polyoxyl 35 castor oil, polyoxyl 40 hydrogenated castor oil, carbomer 1342, corn oil-mono-di-triglyceride, polyoxyethylated oleic acid glyceride, poloxamer, or a combination thereof.

7. The composition of claim 5, wherein the nonionic flocculant is a polysorbate, an alkylphenol ethoxylate, a sorbitan ester, a poloxamer, or a combination thereof.

8. The composition of claim 5, wherein the nonionic flocculant is a polysorbate or a sorbitan ester.

9. The composition of any one of the preceding claims, wherein the concentration of the flocculant in the composition is less than about 10 mg / mL.

10. The composition of any one of the preceding claims, wherein the composition has a flocculated volume of greater than about 60% after mixing.

11. The composition of any of the preceding claims, wherein the flocculated volume decreases by less than about 10% after at least one month of storage in a closed container at less than about 40°C.

12. The composition of any one of the preceding claims, wherein the flocculated volume of the composition is substantially the same for at least one month under container closed storage conditions below about 40°C.

13. The composition of any one of the preceding claims, wherein the plurality of particles and flocculant remain substantially suspended in the liquid carrier for at least one month under container-closed storage conditions below about 40°C.

14. The composition of any of the preceding claims, wherein aggregation of the therapeutic biologic of substantially all of the particles is less than about 10%.

15. The composition of any of the preceding claims, wherein substantially all of the particles are substantially free of any aggregation of the therapeutic biologic.

16. The composition of any of the preceding claims, wherein substantially all of the particles have less than about 10% fragmentation of the therapeutic biologic.

17. The composition of any of the preceding claims, wherein substantially all of the particles are substantially free of any fragmentation of the therapeutic biologic.

18. The composition of any of the preceding claims, wherein substantially all of the particles have a charge variation of the therapeutic biologic of less than about 5%.

19. The composition of any of the preceding claims, wherein substantially all of the particles are substantially free of any change in the charge variant of the therapeutic biologic.

20. The composition of any of the preceding claims, wherein substantially all of the particles have a circularity of from about 0.80 to about 1.

00.

21. The composition of any one of the preceding claims, wherein substantially all of the particles have a circularity of about 1.

00.

22. The composition of any of the preceding claims, wherein substantially all of the particles have a moisture content of less than about 7% by weight.

23. The composition of any of the preceding claims, wherein substantially all of the particles have less than about 10% internal void space.

24. A composition according to any one of the preceding claims, wherein substantially all of the particles are substantially free of any internal void space.

25. The composition of any of the preceding claims, wherein substantially all of the particles have greater than about 60% by weight therapeutic biologic.

26. The composition of any of the preceding claims, wherein substantially all of the particles further comprise a carbohydrate, a pH adjuster, a salt, a chelating agent, a surfactant, a protein stabilizer, an emulsifier, an antimicrobial agent, an amino acid, an antioxidant, a paraben, a bactericide, a fungicide, a preservative, or a combination thereof.

27. The composition of claim 26, wherein the carbohydrate is dextran, trehalose, sucrose, agarose, mannitol, lactose, sorbitol, maltose, or a combination thereof.

28. The composition of claim 26, wherein the pH adjuster is acetate, citrate, glutamate, glycinate, histidine, lactate, maleate, phosphate, succinate, tartrate, bicarbonate, aluminum hydroxide, phosphoric acid, hydrochloric acid, DL-lactic / glycolic acid, phosphoethanolamine, tromethamine, imidazole, glycylglycine, monosodium glutamate, sodium hydroxide, potassium hydroxide, or a combination thereof.

29. The composition of claim 26, wherein the salt is sodium chloride, calcium chloride, potassium chloride, sodium hydroxide, stannous chloride, magnesium sulfate, sodium glucoheptonate, sodium pertechnetate, guanidine hydrochloride, potassium hydroxide, magnesium chloride, potassium nitrate, or a combination thereof.

30. The composition of claim 26, wherein the chelating agent is edetate disodium, ethylenediaminetetraacetic acid, pentetic acid, or a combination thereof.

31. The composition of claim 26, wherein the surfactant is a sorbitan ester, a polysorbate, a poloxamer, polyethylene glycol tert-octylphenyl ether, lecithin, sodium stearate, magnesium stearate, or a combination thereof.

32. The composition of claim 26, wherein the protein stabilizer is trehalose, polyethylene glycol (PEG), poloxamer, polyvinyl pyrrolidone, polyacrylic acid, poly(vinyl) polymer, polyester, polyaldehyde, tertiary polymer, polyamino acid, hydroxyethyl starch, N-methyl-2-pyrrolidone, sorbitol, sucrose, mannitol, cyclodextrin, hydroxypropyl β-cyclodextrin, sulfobutyl ether β-cyclodextrin, methyl β-cyclodextrin, acetyl tryptophan ester, caprylate, N-acetyl tryptophan, propylene glycol, glucose star polymer, silicone polymer, polydimethylsiloxane, carboxymethyl cellulose, poly(glycolic acid), poly(lactic-co-glycolic acid), polylactic acid, polycaprolactone (PCL), polyvinyl pyrrolidone (PVP), polysucrose, dextran, or a combination thereof.

33. The composition of claim 26, wherein the emulsifier is polysorbate, sorbitan monooleate, ethanolamine, polyoxyl 35 castor oil, polyoxyl 40 hydrogenated castor oil, carbomer 1342, corn oil-mono-di-triglyceride, polyoxyethylated oleylglyceride, poloxamer, or a combination thereof.

34. The composition of claim 26, wherein the antimicrobial agent is phenol, m-cresol, benzyl alcohol, 2-phenoxyethanol, chlorobutanol, neomycin, benzethonium chloride, glutaraldehyde, β-propiolactone, or a combination thereof.

35. The composition of claim 26, wherein 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 a combination thereof.

36. The composition of claim 26, wherein the antioxidant is glutathione, ascorbic acid, cysteine, N-acetyl-L-tryptophanate, tocopherol, histidine, methionine, pentetic acid, or a combination thereof.

37. The composition of claim 26, wherein the paraben is a paraben.

38. The composition of claim 26, wherein the antiseptic is benzalkonium chloride.

39. The composition of claim 26, wherein the fungicide is acibenzolar, 2-phenylphenol, anilazine, carvone, natamycin, potassium azide, or a combination thereof.

40. The composition of claim 26, wherein the preservative is sodium nitrate, sulfur dioxide, potassium sorbate, sodium sorbate, sodium benzoate, benzoic acid, methyl hydroxybenzoate, thimerosal, parabens, formaldehyde, castor oil, or a combination thereof.

41. The composition of any one of claims 1 to 25, wherein substantially all of the particles further comprise at least one carbohydrate and an amino acid.

42. A composition according to any one of claims 1 to 25, wherein substantially all of the particles further comprise at least one surfactant and an amino acid.

43. The composition of any one of claims 1 to 25, wherein substantially all of the particles further comprise at least one amino acid.

44. A composition according to any of the preceding claims, wherein the therapeutic biologic is an antibody or fragment thereof.

45. The composition of claim 44, wherein the antibody is a human antibody.

46. ​​The composition of claim 45, wherein the human antibody is an IgG antibody.

47. The composition of any one of claims 44 to 46, wherein the antibody is a monoclonal antibody.

48. A composition according to any one of the preceding claims, wherein the liquid carrier is non-aqueous.

49. The composition of claim 48, wherein the non-aqueous liquid carrier is an organic solvent.

50. The composition according to claim 49, wherein the organic solvent comprises benzyl benzoate, coconut oil, cottonseed oil, fish oil, grape seed 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, dimethyl isosorbide, dimethyl sulfoxide, glycogen, diethylene glycol dimethyl ether, methyl tert-butyl ether, N-methylpyrrolidone, Pyrrolidone, perfluorodecalin, polyethylene glycol, 2-pyrrolidone, tetrahydrofurfuryl alcohol, diglycerides, triglycerides, medium chain triglycerides (MCT), caproic acid, caprylic acid, capric acid, lauric acid, lauryl ethyl ester, fractionated vegetable fatty acid C8 and C10 triglycerides, saturated vegetable fatty acid C8 and C10 propylene glycol diester (PGD), ethyl oleate, ethyl caprate, dibutyl adipate, fatty acid esters, caproic acid, caprylic acid, triacetin, diethylene glycol monoether, γ-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, γ-butyrolactone, tocopherol, octafluoropropane, (perfluorohexyl) octane, n-acetyltryptophan, ethyl laurate, methyl caprylate, ethyl caprylate, methyl caprate, methyl myristic acid, methyl oleate, methyl linoleate, dimethyl adipate, dibutyl suberate, diethyl sebacate, Australia Argan oil ethyl ester, trimethylolpropane triisostearate, isopropyl laurate, isopropyl myristate, diethyl succinate, polysorbate, ethanolamine, propionic acid, citral, anisole, anethole, benzaldehyde, linalool, caprolactone, phenol, thioglycerol, dimethylacetamide, diethylene glycol monoethyl ether, glyceryl acetonate, dimethyl isosorbide, ethyl formate, ethylhexyl acetate, propylene glycol dicaprylate, caprylic triglyceride, ethyl linoleate, ethyl linolenate, or a combination thereof.

51. The composition of claim 50, wherein the organic solvent is ethyl oleate, diglyceride, triglyceride, macadamia oleate, ethyl caprate, diethyl succinate, diethylene glycol monoethyl ether, propylene glycol dicaprylate, caprylic triglyceride, ethyl linoleate, ethyl linolenate, medium chain triglycerides (MCT), sesame oil, propylene glycol diesters of saturated vegetable fatty acids C8 and C10 (PGD), triacetin, or a combination thereof.

52. The composition of claim 50, wherein the organic solvent is ethyl oleate, ethyl caprylate, propylene glycol dicaprylate, diglycerides, triglycerides, sesame oil, propylene glycol diesters (PGD) of saturated vegetable fatty acids C8 and C10, triacetin, or a combination thereof.

53. The composition of claim 48, wherein the non-aqueous liquid carrier comprises at least two organic solvents.

54. The composition of any of the preceding claims, wherein the concentration of the therapeutic biologic in the composition is greater than about 400 mg / mL.

55. The composition of any preceding claim, wherein the composition has a viscosity of less than about 100 mPa·s.

56. A composition according to any one of the preceding claims, wherein the composition is substantially free of visible particles (VP) when dissolved in an aqueous liquid.

57. The composition of any of the preceding claims, wherein the composition has a concentration of insoluble subvisible particles (SvP) having a characteristic size greater than about 10 μm of from about 0 / mL to about 100,000,000 / mL when dissolved in an aqueous liquid.

58. The composition of any of the preceding claims, wherein the composition has a concentration of insoluble subvisible particles (SvP) having a characteristic size greater than about 10 μm from about 0 / mL to about 6000 / mL when dissolved in an aqueous liquid.

59. The composition of any of the preceding claims, wherein the composition has a concentration of insoluble subvisible particles (SvP) having a characteristic size greater than about 25 μm of from about 0 / mL to about 600 / mL when dissolved in an aqueous liquid.

60. The composition of any preceding claim, wherein the composition is substantially free of insoluble subvisible particles (SvP) when dissolved in an aqueous liquid.

61. The composition of any of the preceding claims, wherein the aqueous liquid is water, an aqueous buffer, or a physiologically relevant aqueous liquid.

62. A composition according to any of the preceding claims, wherein the immunogenicity of the composition is substantially similar compared to an aqueous composition comprising at least one therapeutic biologic in soluble form.

63. A composition according to any of the preceding claims, wherein the composition is substantially non-immunogenic.

64. The composition of any of the preceding claims, wherein the toxicity of the composition is substantially similar compared to an aqueous composition comprising at least one therapeutic biologic in a soluble form.

65. The composition of any of the preceding claims, wherein the composition is substantially non-toxic.

66. The composition of any of the preceding claims, wherein the pharmacokinetics (PK) of the composition is improved compared to an aqueous composition comprising at least one therapeutic biologic in a soluble form.

67. The composition of any of the preceding claims, wherein the stability of the therapeutic biologic of the composition is improved compared to an aqueous composition comprising at least one therapeutic biologic in a soluble form.

68. A composition according to any of the preceding claims, wherein the therapeutic biologic in the composition is stable for at least one month.

69. A composition according to any of the preceding claims, wherein the therapeutic biologic in the composition is stable for at least three months.

70. The composition of any of the preceding claims, wherein the therapeutic biologic in the composition is stable for at least three months at 40°C.

71. A method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a pharmaceutically effective amount of a composition comprising: a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein substantially all of the particles comprise at least one therapeutic biologic or a salt thereof; and A flocculant, wherein the concentration of the flocculant in the composition is less than about 50 mg / mL; and wherein the concentration of the therapeutic biologic or salt thereof in the composition is greater than about 250 mg / mL.

72. The method of claim 71, wherein the disease or condition is cancer, an inflammatory disease, or an immune disease.

73. The method of claim 71, wherein substantially all of the particles comprise at least one therapeutic biologic.

74. The method of claim 71, wherein the flocculant is ionic.

75. The method of claim 74, wherein the ionic flocculant is magnesium stearate, sodium lauryl sulfate, sodium stearate, cetyltrimethylammonium bromide, lecithin, or a combination thereof.

76. The method of claim 71, wherein the flocculant is nonionic.

77. The method of claim 76, wherein the nonionic flocculant is polysorbate, alkylphenol ethoxylate, glycerol, polyoxyethylene castor oil, docusate, decyl glucoside, nonoxynol-9, sorbitan esters, sorbitan monooleate, ethanolamine, polyoxyl 35 castor oil, polyoxyl 40 hydrogenated castor oil, carbomer 1342, corn oil-mono-di-triglyceride, polyoxyethylated oleyl glyceride, poloxamer, or a combination thereof.

78. The method of claim 76, wherein the nonionic flocculant is a polysorbate, an alkylphenol ethoxylate, a sorbitan ester, a poloxamer, or a combination thereof.

79. The method of claim 76, wherein the nonionic flocculant is a polysorbate or a sorbitan ester.

80. The method of any one of claims 71 to 79, wherein the concentration of the flocculant in the composition is less than about 10 mg / mL.

81. The method of any one of claims 71 to 80, wherein the composition has a flocculated volume of greater than about 60% after initial mixing.

82. The method of any one of claims 71 to 81, wherein the flocculated volume decreases by less than about 10% after at least one month in a container-closed storage condition below about 40°C.

83. The method of any one of claims 71 to 82, wherein the flocculated volume of the composition is substantially the same for at least one month under container-closed storage conditions below about 40°C.

84. The method of any one of claims 71 to 83, wherein the plurality of particles and flocculant remain substantially suspended in the liquid carrier for at least one month under container-closed storage conditions below about 40°C.

85. The method of any one of claims 71 to 84, wherein substantially all of the particles have less than about 10% aggregation of the therapeutic biologic.

86. The method of any one of claims 71 to 85, wherein substantially all of the particles are substantially free of any aggregation of the therapeutic biologic.

87. The method of any one of claims 71 to 86, wherein substantially all of the particles of the therapeutic biologic have less than about 10% fragmentation.

88. The method of any one of claims 71 to 87, wherein substantially all of the particles are substantially free of any fragmentation of the therapeutic biologic.

89. The method of any one of claims 71 to 88, wherein substantially all of the particles have a charge variation of the therapeutic biologic of less than about 5%.

90. The method of any one of claims 71 to 89, wherein substantially all of the particles are substantially free of any change in the charge variant of the therapeutic biologic.

91. The method of any one of claims 71 to 90, wherein substantially all of the particles have a circularity of from about 0.80 to about 1.

00.

92. The method of any one of claims 71 to 91, wherein substantially all of the particles have a circularity of about 1.

00.

93. The method of any one of claims 71 to 92, wherein substantially all of the particles have a moisture content of less than about 7% by weight.

94. The method of any one of claims 71 to 93, wherein substantially all of the particles have less than about 10% internal void space.

95. The method of any one of claims 71 to 94, wherein substantially all of the particles are substantially free of any internal void space.

96. The method of any one of claims 71 to 95, wherein substantially all of the particles have greater than about 60% by weight therapeutic biologic.

97. The method of any one of claims 71 to 96, wherein substantially all of the particles further comprise a carbohydrate, a pH adjuster, a salt, a chelating agent, a surfactant, a protein stabilizer, an emulsifier, an antimicrobial agent, an amino acid, an antioxidant, a paraben, a bactericide, a fungicide, a preservative, or a combination thereof.

98. The method of claim 97, wherein the carbohydrate is dextran, trehalose, sucrose, agarose, mannitol, lactose, sorbitol, maltose, or a combination thereof.

99. The method of claim 97, wherein the pH adjusting agent is acetate, citrate, glutamate, glycinate, histidine, lactate, maleate, phosphate, succinate, tartrate, bicarbonate, aluminum hydroxide, phosphoric acid, hydrochloric acid, DL-lactic / glycolic acid, phosphoethanolamine, tromethamine, imidazole, glycylglycine, monosodium glutamate, sodium hydroxide, potassium hydroxide, or a combination thereof.

100. The method of claim 97, wherein the salt is sodium chloride, calcium chloride, potassium chloride, sodium hydroxide, stannous chloride, magnesium sulfate, sodium glucoheptonate, sodium pertechnetate, guanidine hydrochloride, potassium hydroxide, magnesium chloride, potassium nitrate, or a combination thereof.

101. The method of claim 97, wherein the chelating agent is edetate disodium, ethylenediaminetetraacetic acid, pentetic acid, or a combination thereof.

102. The method of claim 97, wherein the surfactant is a sorbitan ester, a polysorbate, a poloxamer, polyethylene glycol tert-octylphenyl ether, lecithin, sodium stearate, magnesium stearate, or a combination thereof.

103. The method of claim 97, wherein the protein stabilizer is trehalose, polyethylene glycol (PEG), poloxamer, polyvinyl pyrrolidone, polyacrylic acid, poly(vinyl) polymer, polyester, polyaldehyde, tertiary polymer, polyamino acid, hydroxyethyl starch, N-methyl-2-pyrrolidone, sorbitol, sucrose, mannitol, cyclodextrin, hydroxypropyl β-cyclodextrin, sulfobutyl ether β-cyclodextrin, acetyl tryptophan ester, methyl β-cyclodextrin, caprylate, N-acetyl tryptophan, propylene glycol, glucose star polymer, silicone polymer, polydimethylsiloxane, carboxymethyl cellulose, poly(glycolic acid), poly(lactic-co-glycolic acid), polylactic acid, polycaprolactone (PCL), polyvinyl pyrrolidone (PVP), polysucrose, dextran, or a combination thereof.

104. The method of claim 97, wherein the emulsifier is polysorbate, sorbitan monooleate, ethanolamine, polyoxyl 35 castor oil, polyoxyl 40 hydrogenated castor oil, carbomer 1342, corn oil-mono-di-triglyceride, polyoxyethylated olein, poloxamer, or a combination thereof.

105. The method of claim 97, wherein the antimicrobial agent is phenol, m-cresol, benzyl alcohol, 2-phenoxyethanol, chlorobutanol, neomycin, benzethonium chloride, glutaraldehyde, beta-propiolactone, or a combination thereof.

106. The method of claim 97, wherein 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 a combination thereof.

107. The method of claim 97, wherein the antioxidant is glutathione, ascorbic acid, cysteine, N-acetyl-L-tryptophanate, tocopherol, histidine, methionine, pentetic acid, or a combination thereof.

108. The method of claim 97, wherein the paraben is a paraben.

109. The method of claim 97, wherein the antiseptic is benzalkonium chloride.

110. The method of claim 97, wherein the fungicide is acibenzolar, 2-phenylphenol, caprolactam, carvone, natamycin, potassium azide, or a combination thereof.

111. The method of claim 97, wherein the preservative is sodium nitrate, sulfur dioxide, potassium sorbate, sodium sorbate, sodium benzoate, benzoic acid, methyl hydroxybenzoate, thimerosal, parabens, formaldehyde, castor oil, or a combination thereof.

112. The method of claim 97, wherein substantially all of the particles further comprise at least one carbohydrate and an amino acid.

113. The method of claim 97, wherein substantially all of the particles further comprise at least one surfactant and an amino acid.

114. The method of claim 97, wherein substantially all of the particles further comprise at least one amino acid.

115. The method of any one of claims 71 to 114, wherein the therapeutic biologic is an antibody or fragment thereof.

116. The method of claim 115, wherein the antibody is a human antibody.

117. The method of claim 116, wherein the human antibody is an IgG antibody.

118. The method of claim 115, wherein the antibody is a monoclonal antibody.

119. The method of any one of claims 71 to 118, wherein the liquid carrier is non-aqueous.

120. The method of claim 119, wherein the non-aqueous liquid carrier is an organic solvent.

121. The method of claim 120, wherein the organic solvent comprises 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, dimethyl isosorbide, dimethyl sulfoxide, glycogen, diethylene glycol dimethyl ether, methyl tert-butyl ether, N-methylpyrrolidone, Pyrrolidone, perfluorodecalin, polyethylene glycol, 2-pyrrolidone, tetrahydrofurfuryl alcohol, diglycerides, triglycerides, medium chain triglycerides (MCT), caproic acid, caprylic acid, capric acid, lauric acid, lauryl ethyl ester, fractionated vegetable fatty acid C8 and C10 triglycerides, saturated vegetable fatty acid C8 and C10 propylene glycol diester (PGD), ethyl oleate, ethyl caprate, dibutyl adipate, fatty acid esters, caproic acid, caprylic acid, triacetin, diethylene glycol monoether, γ-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, γ-butyrolactone, tocopherol, octafluoropropane, (perfluorohexyl) octane, n-acetyltryptophan, ethyl laurate, methyl caprylate, ethyl caprylate, methyl caprate, methyl myristic acid, methyl oleate, methyl linoleate, dimethyl adipate, dibutyl suberate, diethyl sebacate, Australia Argan oil ethyl ester, trimethylolpropane triisostearate, isopropyl laurate, isopropyl myristate, diethyl succinate, polysorbate, ethanolamine, propionic acid, citral, anisole, anethole, benzaldehyde, linalool, caprolactone, phenol, thioglycerol, dimethylacetamide, diethylene glycol monoethyl ether, glyceryl acetonate, dimethyl isosorbide, ethyl formate, ethylhexyl acetate, propylene glycol dicaprylate, caprylic triglyceride, ethyl linoleate, ethyl linolenate, or a combination thereof.

122. The method of claim 120, wherein the organic solvent is ethyl oleate, diglyceride, triglyceride, macadamia oleate, ethyl caprate, diethyl succinate, diethylene glycol monoethyl ether, propylene glycol dicaprylate, caprylic triglyceride, ethyl linoleate, ethyl linolenate, medium chain triglycerides (MCT), sesame oil, propylene glycol diesters of saturated vegetable fatty acids C8 and C10 (PGD), triacetin, or a combination thereof.

123. The method of claim 120, wherein the organic solvent is ethyl oleate, ethyl caprylate, propylene glycol dicaprylate, diglycerides, triglycerides, sesame oil, propylene glycol diesters (PGD) of saturated vegetable fatty acids C8 and C10, triacetin, or a combination thereof.

124. The method of claim 119, wherein the non-aqueous liquid carrier comprises at least two organic solvents.

125. The method of any one of claims 71 to 124, wherein the concentration of the therapeutic biologic in the composition is greater than about 400 mg / mL.

126. The method of any one of claims 71 to 125, wherein the viscosity of the composition is less than about 100 mPa·s.

127. The method of any one of claims 71 to 126, wherein the composition is substantially free of visible particles (VP) when dissolved in an aqueous liquid.

128. The method of any one of claims 71 to 127, wherein the composition, when dissolved in an aqueous liquid, has a concentration of insoluble subvisible particles (SvP) having a characteristic size greater than about 10 μm of about 0 / mL to about 100,000,000 / mL.

129. The method of any one of claims 71 to 128, wherein the composition, when dissolved in an aqueous liquid, has a concentration of insoluble subvisible particles (SvP) having a characteristic size greater than about 10 μm of about 0 / mL to about 6000 / mL.

130. The method of any one of claims 71 to 129, wherein the composition, when dissolved in an aqueous liquid, has a concentration of insoluble subvisible particles (SvP) having a characteristic size greater than about 25 μm of about 0 / mL to about 600 / mL.

131. The method of any one of claims 71 to 130, wherein the composition is substantially free of insoluble subvisible particles (SvP) when dissolved in an aqueous liquid.

132. The method of any one of claims 71 to 131, wherein the aqueous liquid is water, an aqueous buffer, or a physiologically relevant aqueous liquid.

133. The method of any one of claims 71 to 132, wherein the immunogenicity of the composition is substantially similar compared to an aqueous composition comprising at least one therapeutic biologic in soluble form.

134. The method of any one of claims 71 to 133, wherein the composition is substantially non-immunogenic.

135. The method of any one of claims 71 to 134, wherein the toxicity of the composition is substantially similar compared to an aqueous composition comprising at least one therapeutic biologic in a soluble form.

136. The method of any one of claims 71 to 135, wherein the composition is substantially non-toxic.

137. The method of any one of claims 71 to 136, wherein the pharmacokinetics (PK) of the composition is improved compared to an aqueous composition comprising at least one therapeutic biologic in a soluble form.

138. The method of any one of claims 71 to 137, wherein the method further comprises administering at least one hyaluronan degrading agent.

139. The method of claim 138, wherein the hyaluronan degrading agent is a hyaluronidase.

140. The method of claim 139, wherein the hyaluronidase is a soluble neutral active hyaluronidase.

141. The method of claim 139, wherein the hyaluronidase is a mammalian hyaluronidase.

142. The method of claim 141, wherein the mammalian hyaluronidase is a human hyaluronidase.

143. The method of claim 142, wherein the human hyaluronidase is recombinant human hyaluronidase (rHuPH20).

144. The method of claim 138, wherein the hyaluronan degrading agent is administered simultaneously, sequentially or intermittently with the composition.

145. The method of any one of claims 71 to 144, wherein the composition is administered to a subject in need thereof by parenteral, subcutaneous, oral, epidermal, intradermal, intramuscular, intraarterial, intraperitoneal, or intravenous injection.

146. The method of any one of claims 71 to 145, wherein the composition is administered by subcutaneous injection.

147. The method of any one of claims 71 to 145, wherein the composition is administered by a syringe or a portable drug delivery injection device.

148. The method of claim 147, wherein the syringe or portable drug delivery injection device is pre-filled with the composition.

149. The method of claim 147, wherein the composition is administered by syringe.

150. The method of claim 147, wherein the composition is administered via a portable drug delivery injection device.

151. The method of claim 148, wherein the portable drug delivery injection device is a pen injector.

152. The method of claim 148, wherein the portable drug delivery injection device is an autoinjector.

153. The method of claim 148, wherein the portable drug delivery injection device is an oral administration liquid syringe capsule.

154. The method of any one of claims 71 to 153, wherein the composition is dispensed from a needle having a gauge ranging from 18 to 33 gauge.

155. The method of any one of claims 71 to 154, wherein the composition is dispensed using an injection force of less than about 70N.

156. The method of any one of claims 71 to 155, wherein the injection force remains substantially the same for at least one month under container closed storage conditions below about 40°C.

157. The method of any one of claims 71 to 156, wherein less than about 20.0 mL of the composition to be administered.

158. The method of any one of claims 71 to 157, wherein less than about 2.0 mL of the composition is administered.

159. The method of any one of claims 71 to 158, wherein less than about 0.5 mL of the composition is administered.

160. The method of any one of claims 71 to 159, wherein the composition dissolves in less than about 10 minutes after administration.

161. The method of any one of claims 71 to 160, wherein the composition dissolves immediately after administration.

162. The method of any one of claims 71 to 161, wherein the composition is administered in one or more doses.

163. The method of claim 162, wherein the composition is administered as a single dose.

164. The method of claim 162, wherein the composition is administered in multiple doses.

165. The method of any one of claims 71 to 164, wherein the subject is a mammal.

166. The method of any one of claims 71 to 165, wherein the subject is a human.

167. The method of any one of claims 71 to 166, wherein the composition has improved stability of the therapeutic biologic compared to an aqueous composition comprising at least one therapeutic biologic in a soluble form.

168. The method of any one of claims 71 to 167, wherein the therapeutic biologic in the composition is stable for at least one month.

169. The method of any one of claims 71 to 168, wherein the therapeutic biologic in the composition is stable for at least three months.

170. The method of any one of claims 71 to 169, wherein the therapeutic biologic in the composition is stable at 40°C for at least three months.

171. A method of administering a pharmaceutically effective composition, the method comprising: a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein substantially all of the particles comprise at least one therapeutic biologic or a salt thereof; and A flocculant, wherein the concentration of the flocculant in the composition is less than about 50 mg / mL; and wherein the concentration of the therapeutic biologic or salt thereof in the composition is greater than about 250 mg / mL.

172. The method of claim 171, wherein substantially all of the particles comprise at least one therapeutic biologic.

173. The method of claim 171, wherein the flocculant is ionic.

174. The method of claim 173, wherein the ionic flocculant is magnesium stearate, sodium lauryl sulfate, sodium stearate, cetyltrimethylammonium bromide, lecithin, or a combination thereof.

175. The method of claim 171, wherein the flocculant is non-ionic.

176. The method of claim 175, wherein the nonionic flocculant is polysorbate, alkylphenol ethoxylate, glycerol, polyoxyethylene castor oil, docusate, decyl glucoside, nonoxynol-9, sorbitan esters, sorbitan monooleate, ethanolamine, polyoxyl 35 castor oil, polyoxyl 40 hydrogenated castor oil, carbomer 1342, corn oil-mono-di-triglyceride, polyoxyethylated oleic acid glyceride, poloxamer, or a combination thereof.

177. The method of claim 175, wherein the nonionic flocculant is a polysorbate, an alkylphenol ethoxylate, a sorbitan ester, a poloxamer, or a combination thereof.

178. The method of claim 175, wherein the nonionic flocculant is a polysorbate or a sorbitan ester.

179. The method of any one of claims 171 to 178, wherein the concentration of the flocculant in the composition is less than about 10 mg / mL.

180. The method of any one of claims 171 to 179, wherein the composition has a flocculated volume of greater than about 60% after mixing.

181. The method of any one of claims 171 to 180, wherein the flocculated volume decreases by less than about 10% after at least one month in a container-closed storage condition below about 40°C.

182. The method of any one of claims 171 to 181, wherein the flocculated volume of the composition is substantially the same for at least one month under container-closed storage conditions below about 40°C.

183. The method of any one of claims 171 to 182, wherein the plurality of particles and flocculant remain substantially suspended in the liquid carrier for at least one month under container-closed storage conditions below about 40°C.

184. The method of any one of claims 171 to 183, wherein aggregation of the therapeutic biologic of substantially all of the particles is less than about 10%.

185. The method of any one of claims 171 to 184, wherein substantially all of the particles are substantially free of any aggregation of the therapeutic biologic.

186. The method of any one of claims 171 to 185, wherein substantially all of the particles of the therapeutic biologic have less than about 10% fragmentation.

187. The method of any one of claims 171 to 186, wherein substantially all of the particles are substantially free of any fragmentation of the therapeutic biologic.

188. The method of any one of claims 171 to 187, wherein substantially all of the particles have a charge variant of the therapeutic biologic that varies by less than about 5%.

189. The method of any one of claims 171 to 188, wherein substantially all of the particles are substantially free of any change in the charge variant of the therapeutic biologic.

190. The method of any one of claims 171 to 189, wherein substantially all of the particles have a circularity of about 0.80 to about 1.

00.

191. The method of any one of claims 171 to 190, wherein substantially all of the particles have a circularity of about 1.

00.

192. The method of any one of claims 171 to 191, wherein substantially all of the particles have a moisture content of less than about 7% by weight.

193. The method of any one of claims 171 to 192, wherein substantially all of the particles have less than about 10% internal void space.

194. The method of any one of claims 171 to 193, wherein substantially all of the particles are substantially free of any internal void space.

195. The method of any one of claims 171 to 194, wherein substantially all of the particles have greater than about 60% by weight therapeutic biologic.

196. The method of any one of claims 171 to 195, wherein substantially all of the particles further comprise a carbohydrate, a pH adjuster, a salt, a chelating agent, a surfactant, a protein stabilizer, an emulsifier, an antimicrobial agent, an amino acid, an antioxidant, a paraben, a bactericide, a fungicide, a preservative, or a combination thereof.

197. The method of any one of claims 171 to 195, wherein substantially all of the particles further comprise at least one carbohydrate and an amino acid.

198. The method of any one of claims 171 to 195, wherein substantially all of the particles further comprise at least one surfactant and an amino acid.

199. The method of any one of claims 171 to 195, wherein substantially all of the particles further comprise at least one amino acid.

200. The method of any one of claims 171 to 199, wherein the therapeutic biologic is an antibody or fragment thereof.

201. The method of claim 200, wherein the antibody is a human antibody.

202. The method of claim 201, wherein the human antibody is an IgG antibody.

203. The method of any one of claims 200, 201 or 202, wherein the antibody is a monoclonal antibody.

204. The method of any one of claims 171 to 203, wherein the liquid carrier is non-aqueous.

205. The method of claim 204, wherein the non-aqueous liquid carrier is an organic solvent.

206. The method of claim 205, wherein the organic solvent comprises 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, dimethyl isosorbide, dimethyl sulfoxide, glycogen, diethylene glycol dimethyl ether, methyl tert-butyl ether, N-methylpyrrolidone, Pyrrolidone, perfluorodecalin, polyethylene glycol, 2-pyrrolidone, tetrahydrofurfuryl alcohol, diglycerides, triglycerides, medium chain triglycerides (MCT), caproic acid, caprylic acid, capric acid, lauric acid, lauryl ethyl ester, fractionated vegetable fatty acid C8 and C10 triglycerides, saturated vegetable fatty acid C8 and C10 propylene glycol diester (PGD), ethyl oleate, ethyl caprate, dibutyl adipate, fatty acid esters, caproic acid, caprylic acid, triacetin, diethylene glycol monoether, γ-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, γ-butyrolactone, tocopherol, octafluoropropane, (perfluorohexyl) octane, n-acetyltryptophan, ethyl laurate, methyl caprylate, ethyl caprylate, methyl caprate, methyl myristic acid, methyl oleate, methyl linoleate, dimethyl adipate, dibutyl suberate, diethyl sebacate, Australia Argan oil ethyl ester, trimethylolpropane triisostearate, isopropyl laurate, isopropyl myristate, diethyl succinate, polysorbate, ethanolamine, propionic acid, citral, anisole, anethole, benzaldehyde, linalool, caprolactone, phenol, thioglycerol, dimethylacetamide, diethylene glycol monoethyl ether, glyceryl acetonate, dimethyl isosorbide, ethyl formate, ethylhexyl acetate, propylene glycol dicaprylate, caprylic triglyceride, ethyl linoleate, ethyl linolenate, or a combination thereof.

207. The method of claim 204, wherein the non-aqueous liquid carrier comprises at least two organic solvents.

208. The method of any one of claims 171 to 207, wherein the concentration of the therapeutic biologic in the composition is greater than about 400 mg / mL.

209. The method of any one of claims 171 to 208, wherein the viscosity of the composition is less than about 100 mPa·s.

210. The method of any one of claims 171 to 209, wherein the composition is substantially free of visible particles (VP) when dissolved in an aqueous liquid.

211. The method of any one of claims 171 to 210, wherein the composition, when dissolved in an aqueous liquid, has a concentration of insoluble subvisible particles (SvP) having a characteristic size greater than about 10 μm of about 0 / mL to about 100,000,000 / mL.

212. The method of any one of claims 171 to 211, wherein the composition, when dissolved in an aqueous liquid, has a concentration of insoluble subvisible particles (SvP) having a characteristic size greater than about 10 μm of about 0 / mL to about 6000 / mL.

213. The method of any one of claims 171 to 212, wherein the composition, when dissolved in an aqueous liquid, has a concentration of insoluble subvisible particles (SvP) having a characteristic size greater than about 25 μm of about 0 / mL to about 600 / mL.

214. The method of any one of claims 171 to 213, wherein the composition is substantially free of insoluble subvisible particles (SvP) when dissolved in an aqueous liquid.

215. The method of any one of claims 171 to 214, wherein the aqueous liquid is water, an aqueous buffer, or a physiologically relevant aqueous liquid.

216. The method of any one of claims 171 to 215, wherein the immunogenicity of the composition is substantially similar compared to an aqueous composition comprising at least one therapeutic biologic in soluble form.

217. The method of any one of claims 171 to 216, wherein the composition is substantially non-immunogenic.

218. The method of any one of claims 171 to 217, wherein the toxicity of the composition is substantially similar compared to an aqueous composition comprising at least one therapeutic biologic in a soluble form.

219. The method of any one of claims 171 to 218, wherein the composition is substantially non-toxic.

220. The method of any one of claims 171 to 219, wherein the pharmacokinetics (PK) of the composition is improved compared to an aqueous composition comprising at least one therapeutic biologic in a soluble form.

221. The method of any one of claims 171 to 220, further comprising administering at least one hyaluronan degrading agent.

222. The method of claim 221, wherein the hyaluronan degrading agent is a hyaluronidase.

223. The method of claim 221, wherein the hyaluronan degrading agent is administered simultaneously, sequentially or intermittently with the composition.

224. The method of any one of claims 171 to 223, wherein the composition is administered to a subject in need thereof by parenteral, subcutaneous, oral, epidermal, intradermal, intramuscular, intraarterial, intraperitoneal, or intravenous injection.

225. The method of any one of claims 171 to 224, wherein the composition is administered by subcutaneous injection.

226. The method of any one of claims 171 to 225, wherein the composition is administered by a syringe or a portable drug delivery injection device.

227. The method of claim 226, wherein the syringe or portable drug delivery injection device is pre-filled with the composition.

228. The method of claim 226, wherein the composition is administered by syringe.

229. The method of claim 226, wherein the composition is administered via a portable drug delivery injection device.

230. The method of claim 229, wherein the portable drug delivery injection device is a pen injector.

231. The method of claim 229, wherein the portable drug delivery injection device is an autoinjector.

232. The method of claim 229, wherein the portable drug delivery injection device is an oral administration liquid syringe capsule.

233. The method of any one of claims 171 to 232, wherein the composition is dispensed from a needle having a gauge ranging from 18 to 33 gauge.

234. The method of any one of claims 171 to 233, wherein the composition is dispensed using an injection force of less than about 70N.

235. The method of any one of claims 171 to 234, wherein the injection force remains substantially the same for at least one month under container closed storage conditions below about 40°C.

236. The method of any one of claims 171 to 235, wherein less than about 20.0 mL of the composition to be administered.

237. The method of any one of claims 171 to 236, wherein less than about 2.0 mL of the composition to be administered.

238. The method of any one of claims 171 to 237, wherein less than about 0.5 mL of the composition to be administered.

239. The method of any one of claims 171 to 238, wherein the composition dissolves in less than about 10 minutes after administration.

240. The method of any one of claims 171 to 239, wherein the composition dissolves immediately after administration.

241. The method of any one of claims 171 to 240, wherein the composition is administered in one or more doses.

242. The method of any one of claims 171 to 241, wherein the subject is a mammal.

243. The method of any one of claims 171 to 242, wherein the subject is a human.

244. The method of any one of claims 171 to 243, wherein the composition has improved stability of the therapeutic biologic compared to an aqueous composition comprising at least one therapeutic biologic in a soluble form.

245. The method of any one of claims 171 to 244, wherein the therapeutic biologic in the composition is stable for at least one month.

246. The method of any one of claims 171 to 245, wherein the therapeutic biologic in the composition is stable for at least three months.

247. The method of any one of claims 171 to 246, wherein the therapeutic biologic in the composition is stable at 40°C for at least three months.

248. A pharmaceutically effective composition comprising particles suspended in a pharmaceutically acceptable liquid carrier, wherein the particles comprise: a first plurality of particles, the first plurality of particles comprising a first therapeutic biologic or a salt thereof; a second plurality of particles comprising a second therapeutic biologic or a salt thereof; and a flocculant, wherein the concentration of the flocculant in the composition is less than about 50 mg / mL; wherein the concentration of the first therapeutic biologic and the second therapeutic biologic or salt thereof in the composition is greater than about 250 mg / mL.

249. The composition of claim 248, wherein the first plurality of particles does not comprise the second therapeutic biologic.

250. The composition of claim 248 or 249, wherein the second plurality of particles does not comprise the first therapeutic biologic.

251. The composition of any one of claims 248 to 250, wherein the first therapeutic biologic is rituximab.

252. The composition of any one of claims 248 to 251, wherein the second therapeutic biologic is trastuzumab.

253. A kit comprising: a syringe or a portable drug delivery injection device; and a composition comprising: a plurality of particles suspended in a pharmaceutically acceptable liquid carrier, wherein substantially all of the particles comprise at least one therapeutic biologic or a salt thereof; and Flocculants, wherein the concentration of the flocculant in the composition is less than about 50 mg / mL, and wherein the concentration of the therapeutic biologic or salt thereof in the composition is greater than about 250 mg / mL.

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