Polypeptide Preparations

Abstract

The present disclosure provides, inter alia, methods and compositions for oral administration of polypeptides. Typically, many polypeptides are administered in solution by intravenous or subcutaneous injection. The present disclosure provides methods and compositions, including polypeptide formulations for oral delivery, including a core and a pharma- ceutically acceptable capsule, where the core includes an amorphous or crystallized polypeptide composition and a pharma-ceutically acceptable carrier that includes a vitamin E agent. The present disclosure includes the surprising discovery that by orally administering a formulation that includes the polypeptide and vitamin E disclosed herein, the polypeptide is successfully delivered to the bloodstream.

Description

[Background technology]

[0001] Polypeptide therapeutics play an important role in medicine. Many modern therapeutics contain polypeptides such as enzymes and antibodies. The typical route of administration of polypeptide therapeutics is intravenous or subcutaneous injection. Injecting therapeutics has well-known drawbacks. Researchers have long sought alternative compositions and methods for administration of polypeptide therapeutics. Summary of the Invention

[0002] The present disclosure provides, inter alia, methods and compositions for administration (e.g., oral administration) of polypeptides. Typically, many polypeptides are formulated as solutions for administration by intravenous or subcutaneous injection. The present disclosure provides, inter alia, encapsulated polypeptide formulations and suspensions for oral delivery, comprising an amorphous or crystallized polypeptide composition (e.g., a powder form comprising an amorphous or crystallized polypeptide) and a pharma- ceutically acceptable carrier comprising a vitamin E agent. The present disclosure includes the surprising discovery that oral administration of a formulation comprising a polypeptide and vitamin E disclosed herein successfully delivers the polypeptide to the bloodstream. Without wishing to be bound by any particular scientific theory, the present disclosure further includes the surprising discovery that a polypeptide orally administered in a formulation disclosed herein comprising a vitamin E agent is efficiently delivered to the bloodstream, lymphatic system, and thoracic duct. In various embodiments, a polypeptide orally administered in a formulation disclosed herein comprising a vitamin E agent is efficiently delivered to the bloodstream via the lymphatic system (e.g., at least partially via the thoracic duct). The present disclosure includes the recognition that the formulations provided herein, including a polypeptide and vitamin E, provide a versatile platform due to their general ability to deliver polypeptides to the bloodstream. Thus, one skilled in the art will understand from this disclosure (including the present examples) that the formulations provided herein are advantageous and useful without the need to specify the polypeptide contained in the formulation. Still further, the present disclosure demonstrates unexpectedly advantageous properties of various oral delivery of the formulations disclosed herein, including improved pharmacokinetic properties, e.g., increased delivery rate and / or half-life to the bloodstream.

[0003] In at least one embodiment, the disclosure includes a polypeptide formulation for oral delivery comprising (i) a crystallized or amorphous polypeptide composition and (ii) a pharma- ceutically acceptable carrier comprising a vitamin E agent, optionally wherein (i) the polypeptide formulation is an encapsulated polypeptide formulation comprising a core and a pharma- ceutically acceptable capsule, wherein the core comprises a crystallized or amorphous polypeptide composition and a pharma- ceutically acceptable carrier, or (ii) the polypeptide formulation is a suspension formulation.

[0004] In at least one aspect, the present disclosure comprises a method for delivering polypeptide to the bloodstream of a subject, comprising orally administering to a subject a polypeptide formulation for oral delivery, comprising (i) a crystallized or amorphous polypeptide composition and (ii) a pharmacologic carrier comprising a vitamin E agent, and optionally (i) the polypeptide formulation is an encapsulated polypeptide formulation comprising a core and a pharmacologic acceptable capsule, wherein the core comprises a crystallized or amorphous polypeptide composition and a pharmacologic acceptable carrier, or (ii) the polypeptide formulation is a suspension formulation.In certain embodiments, the polypeptide is delivered to the bloodstream via the lymphatic system, and optionally the polypeptide is delivered to the bloodstream via the thoracic duct.

[0005] In at least one embodiment, the disclosure includes a method of delivering a polypeptide to the thoracic duct or lymph of a subject, the method comprising orally administering to the subject a polypeptide formulation for oral delivery comprising (i) a crystallized or amorphous polypeptide composition and (ii) a pharma- ceutical acceptable carrier comprising a vitamin E agent, optionally wherein (i) the polypeptide formulation is an encapsulated polypeptide formulation comprising a core and a pharma- ceutical acceptable capsule, the core comprising the crystallized or amorphous polypeptide composition and a pharma- ceutical acceptable carrier, or (ii) the polypeptide formulation is a suspension formulation.

[0006] In at least one embodiment, the disclosure includes a method of producing an encapsulated polypeptide formulation for oral delivery, the method comprising encapsulating within a capsule a core comprising (i) an amorphous or crystallized polypeptide composition and (ii) a pharma- ceutically acceptable carrier comprising a vitamin E agent. In at least one embodiment, the disclosure includes a method of producing a polypeptide suspension for oral delivery, the method comprising suspending (i) a crystallized or amorphous polypeptide composition in (ii) a pharma-ceutically acceptable carrier comprising a vitamin E agent.

[0007] In certain embodiments, the polypeptide comprises (i) a therapeutic polypeptide, (ii) an antibody drug or fragment thereof, (iii) a monoclonal antibody or fragment thereof, (iv) a fusion polypeptide, (v) an immunoglobulin, (vi) an enzyme, or (vii) an analog and / or modified form of any of (i)-(vi), optionally wherein the polypeptide is modified by one or more of pegylation, acetylation, amidation, lipidation, methylation, phosphorylation, glycosylation, glycation, sulfation, mannosylation, nitrosylation, acylation, palmitoylation, prenylation, fatty acids, or combinations thereof. In certain embodiments, the polypeptide is an antibody (e.g., an anti-HER2 antibody, e.g., trastuzumab), a GLP-1 receptor agonist, human glucagon-like peptide-1 (GLP-1) or an analog thereof (e.g., a synthetic analog, e.g., liraglutide), a parathyroid hormone (e.g., a recombinant human parathyroid hormone analog, e.g., teriparatide), insulin (e.g., Humulin®), rituximab, bevacizumab, cetuximab, etanercept, infliximab, agalsidase beta, agalsidase alpha, imiglucerase, aliglucerase alpha, velaglucerase alpha, alglucerase, sebelipase alpha, laronidase, izulus In some embodiments, the polypeptide is selected from Factor I, Factor II, Factor III, Factor IV, Factor V, Factor VI, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, Factor XIII, von Willebrand factor, etanercept, aflibercept, rilonacept, alefacept, romiplostim, abatacept / belatacept, and / or denileukin-diftitox, or an analog or derivative thereof, and optionally the polypeptide is natural, synthetic or engineered.In certain embodiments, the polypeptide has a molecular weight between about 50 Da and about 25 kDa, optionally between about 50 Da and about 1 kDa, between about 50 Da and about 2 kDa, between about 50 Da and about 3 kDa, between about 50 Da and about 4 kDa, between about 50 Da and about 5 kDa, between about 50 Da and about 10 kDa, between about 50 Da and about 15 kDa, or between about 50 Da and about 20 kDa. In certain embodiments, the polypeptide has a molecular weight between about 25 kDa and about 1,000 kDa, optionally between about 25 kDa and about 500 kDa, between about 100 kDa and about 500 kDa, between about 120 kDa and about 250 kDa, or between about 150 kDa and about 300 kDa. In certain embodiments, the polypeptide formulation comprises from about 1 μg to about 2,000 mg of the polypeptide, and optionally the polypeptide formulation comprises from about 1 μg to about 1,000 mg, from about 1 μg to about 500 mg, from about 1 μg to about 400 mg, from about 1 μg to about 300 mg, from about 1 μg to about 200 mg, from about 1 μg to about 100 mg, from about 1 μg to about 50 mg, from about 1 μg to about 25 mg, from about 1 μg to about 20 mg, from about 1 μg to about 15 mg, from about 1 μg to about 10 mg, from about 1 μg to about 1 μg~about 5mg, about 1μg~about 1mg, about 1μg~about 500μg, about 1μg~about 250μg, about 1μg~about 200μg, about 1μg~about 150μg, about 1μg~about 100μg, about 1μg~about 50μg, about 1mg~about 1, 000mg, about 1mg to about 500mg, about 1mg to about 400mg, about 1mg to about 300mg, about 1mg to about 200mg, about 1mg to about 100mg, about 1mg to about 50mg, about 1mg to about 25mg of the polypeptide.

[0008] In certain embodiments, the polypeptide formulation comprises from about 1 mg to about 2,000 mg of the vitamin E agent, optionally the polypeptide formulation comprises from about 1 mg to about 1,000 mg, from about 1 mg to about 500 mg, from about 1 mg to about 400 mg, from about 1 mg to about 300 mg, from about 1 mg to about 200 mg, from about 1 mg to about 100 mg, from about 1 mg to about 50 mg, or from about 1 mg to about 25 mg of the vitamin E agent. In certain embodiments, the polypeptide formulation comprises a molar or weight excess of the vitamin E agent relative to the amount of polypeptide in the polypeptide formulation, optionally the excess is at least a 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, or 1,000-fold excess.

[0009] In certain embodiments, the crystallized polypeptide composition comprises polypeptide crystals having an average particle size of less than 25 microns, e.g., less than 20, 15, 10, 5, 4, 3, 2, 1, or 0.5 microns, e.g., optionally, the polypeptide crystals have an average particle size of between 0.5 microns and 1, 2, 3, 4, 5, 10, 15, 20, or 25 microns, or the polypeptide crystals have an average particle size of between 1 micron and 1, 2, 3, 4, 5, 10, 15, 20, or 25 microns. In certain embodiments, an amorphous polypeptide composition comprises crystals of a polypeptide having an average particle size of less than 25 microns, e.g., less than 20, 15, 10, 5, 4, 3, 2, 1, or 0.5 microns, optionally wherein the polypeptide particles have an average particle size of between 0.5 microns and 1, 2, 3, 4, 5, 10, 15, 20, or 25 microns, or wherein the polypeptide particles have an average particle size of between 1 micron and 1, 2, 3, 4, 5, 10, 15, 20, or 25 microns.

[0010] In certain embodiments, the polypeptide composition comprises a lyophilized polypeptide. In certain embodiments, the polypeptide composition comprises microcrystals of the polypeptide. In certain embodiments, the polypeptide composition comprises a powder comprising a crystallized polypeptide.

[0011] In certain embodiments, the core is a viscous solution. In certain embodiments, the capsule is a hard shell capsule or a soft shell capsule, and optionally, the capsule is a gelatin capsule or an HPMC capsule. In certain embodiments, the capsule is formulated for intestinal delivery, and optionally, the capsule is formulated for stomach and / or intestinal delivery. In certain embodiments, the capsule comprises an enteric coating.

[0012] In certain embodiments, the core further comprises one or more excipients or additives selected from the group consisting of an aggregation reducing agent, a sugar or sugar alcohol, a polysaccharide, a stabilizer, a hyaluronidase, a buffering agent, a preservative, a carrier, an antioxidant, a chelating agent, a natural or synthetic polymer, a cryoprotectant, a lyoprotectant, a surfactant, a bulking agent, an acidifying agent, a component that reduces injection site discomfort, an antifoaming agent, a chelating agent, a natural or synthetic polymer, a cryoprotectant, a cryoprotectant, a surfactant, a bulking agent, an acidifying agent, a component that reduces injection site discomfort, an antifoaming agent, an alkalizing agent, a vehicle, an aggregation inhibitor, a solubilizing agent, a tonicity adjusting agent, and a stabilizer, and combinations thereof. In certain embodiments, the one or more excipients or additives are present, individually or cumulatively, at a concentration of between 0.1 mM and about 1,000 mM, between about 0.1 mM and about 500 mM, between about 0.1 mM and about 200 mM, or between about 0.1 mM and about 100 mM. In certain embodiments, (i) the aggregation reducing agent(s) is selected from the group consisting of nicotinic acid, caffeine citrate, caffeine nicotinate, caffeine, octyl-β-D-glucopyranoside, and n-dodecyl-β-D-maltoside, optionally with arginine, tryptophan, histidine, proline, cysteine, methionine, β-alanine, potassium glutamate, arginine ethyl ester, lysine, aspartic acid, glutamic acid, glycine, DTPA (diethyl ester), glycine, glycerin, glycerol ... Trimethylamine pentaacetic acid), EGTA (aminopolycarboxylic acid), EDTA (ethylenediaminetetraacetic acid), hydroxypropyl beta (HP-beta) cyclodextrin, hydroxypropyl gamma (HP-gamma) cyclodextrin, sulfobutyl ether (SBE) cyclodextrin, TMAO (trimethylamine N-oxide) trehalose, ethylene glycol, betaine, xylitol, sorbitol, 6-(N-(7-nitrobenzo-2-oxa-1,(ii) the tonicity adjusting agent(s) is selected from the group consisting of arginine, cysteine, histidine, glycine, sodium chloride, potassium chloride, sodium citrate, a sugar (e.g., sucrose, glucose, dextrose, glycerin, or mannitol), and a combination thereof; and (iii) the antioxidant(s) is selected from the group consisting of glycine, lysine, EDTA, DTPA, sorbitol, mannitol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, formaldehyde, ethyl cellulose ... or (iv) the lyoprotectant(s) is selected from the group consisting of sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, maltose, lactulose, maltulose, glucitol, maltitol, lactitol, isomaltulose, and mannitol; amino acids (e.g., arginine, histidine, proline, or glycine); lyotropic salts (e.g., magnesium sulfate); propylene glycol, glycerol, poly(ethylene glycol), or poly(propylene glycol); gelatin, dextrin, modified starch, carboxymethylcellulose, and combinations thereof.

[0013] definition a, an, the: As used herein, the articles "a", "an" and "the" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" discloses embodiments of exactly one element as well as embodiments containing two or more elements.

[0014] About: As used herein, the term "about" when used in reference to a value refers to a value similar in context to the referenced value. Generally, a person skilled in the art familiar with the context will understand the appropriate degree of variation that "about" encompasses in that context. For example, in some embodiments, the term "about" can encompass a range of values ​​within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referenced value.

[0015] Administration: As used herein, the term "administration" typically refers to administering a composition to a subject or system to effect delivery of an agent that is the composition itself or that is contained in the composition.

[0016] Agent: As used herein, the term "agent" can refer to any chemical entity, including, but not limited to, any one or more of an atom, a molecule, a compound, an amino acid, a polypeptide, a nucleotide, a nucleic acid, a polypeptide complex, a liquid, a solution, a sugar, a polysaccharide, a lipid, or a combination or complex thereof.

[0017] Amino Acid: As used herein in the broadest sense, refers to any compound and / or substance that can be incorporated into a polypeptide chain, for example, through the formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H 2The amino acid has NC(H)(R)-COOH. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a non-natural amino acid. In some embodiments, the amino acid is a D-amino acid. In some embodiments, the amino acid is an L-amino acid. A "standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. A "non-standard amino acid" refers to an amino acid other than a standard amino acid, whether prepared synthetically or obtained from a natural source. In some embodiments, an amino acid (including the carboxy-terminal amino acid and / or the amino-terminal amino acid in a polypeptide) may contain structural modifications compared to a typical or canonical amino acid structure. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of an amino group, a carboxylic acid group, one or more protons, and / or a hydroxyl group) compared to the general structure. In some embodiments, such modifications may, for example, alter the circulating half-life of a polypeptide comprising the modified amino acid compared to one comprising an otherwise identical unmodified amino acid. In some embodiments, such modifications do not significantly alter the relevant activity of a polypeptide containing the modified amino acid compared to one containing an otherwise identical unmodified amino acid. As will be clear from the context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid, and in some embodiments, to an amino acid residue of a polypeptide.

[0018] Amorphous: As used herein, the term "amorphous" generally refers to a non-crystalline solid form of a polypeptide, sometimes also referred to as an "amorphous solid" or "amorphous precipitate", which typically does not possess, or essentially does not possess, the molecular lattice structure characteristic of the crystalline solid state.

[0019] Analog: As used herein, the term "analog" refers to a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance. Typically, an "analog" exhibits significant structural similarity with a reference substance, e.g., sharing a core or consensus structure, but differs in certain individual respects. In some embodiments, an analog is a substance that can be generated from a reference substance, e.g., by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be generated through the implementation of a synthetic process that is substantially similar (e.g., shares multiple steps) with the process that generates the reference substance. In some embodiments, an analog is generated or can be generated through the implementation of a synthetic process that is different from the process used to generate the reference substance.

[0020] Antibody: As used herein, the term "antibody" refers to a polypeptide that contains one or more canonical immunoglobulin sequence elements (e.g., heavy chain variable domain, light chain variable domain, and / or one or more CDRs) sufficient to confer specific binding to a particular antigen. Thus, the term antibody includes, but is not limited to, human antibodies, non-human antibodies, synthetic antibodies, and / or engineered antibodies, fragments thereof, and agents containing the same. Antibodies are naturally occurring immunoglobulins (e.g., those produced by an organism in response to an antigen). Synthetic, non-natural, or engineered antibodies can be produced by recombinant engineering, chemical synthesis, or other artificial systems or methodologies known to those of skill in the art.

[0021] As is well known in the art, a typical human immunoglobulin is a tetramer of approximately 150 kD containing two identical heavy (H) chain polypeptides (about 50 kD each) and two identical light (L) chain polypeptides (about 25 kD each) that associate with each other to form a structure commonly referred to as a "Y-shape". Typically, each heavy chain contains a heavy chain variable domain (VH) and a heavy chain constant domain (CH). The heavy chain constant domain contains three CH domains: CH1, CH2, and CH3. A short region known as a "switch" connects the heavy chain variable and constant regions. A "hinge" connects the CH2 and CH3 domains to the rest of the immunoglobulin. Each light chain contains a light chain variable domain (VL) and a light chain constant domain (CL), separated from each other by another "switch". Each variable domain contains three hypervariable loops known as "complementarity determining regions" (CDR1, CDR2, and CDR3) and four more or less invariant "framework" regions (FR1, FR2, FR3, and FR4). In each VH and VL, the three CDRs and four FRs are arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Typically, the variable regions of the heavy and / or light chains are understood to provide a binding moiety capable of interacting with an antigen. The constant domains can mediate the binding of the antibody to various immune system cells (e.g., effector cells and / or cells mediating cytotoxicity), receptors, and elements of the complement system. The heavy and light chains are linked to each other by one disulfide bond, and two other disulfide bonds connect the hinge regions of the heavy chains to each other, connecting the dimers to each other to form a tetramer. When native immunoglobulins fold, the FR regions form beta sheets that provide a structural framework for the domain, and the CDR loop regions from both the heavy and light chains come together in three dimensional space to generate a single hypervariable antigen binding site located at the tip of a Y structure.

[0022] In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a monospecific antibody, or a multispecific antibody (including a bispecific antibody). In some embodiments, the antibody comprises at least one light chain monomer or dimer, at least one heavy chain monomer or dimer, at least one heavy chain-light chain dimer, or a tetramer comprising two heavy chain monomers and two light chain monomers. Furthermore, the term "antibody" can include (unless otherwise specified or clear from the context) any art-known construct or format that utilizes the structural and / or functional characteristics of antibodies, including, but not limited to, Zybodies®, Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, isolated CDRs or sets thereof, single chain antibodies, single chain Fvs (scFvs), disulfide-linked Fvs (sdFvs), polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies or fragments thereof, such as IgNAR), camelid antibodies, camelized antibodies, shielding antibodies (e.g., Probodies®), affibodies, anti-idiota Antibody types include, but are not limited to, antibody types (anti-Id) antibodies (e.g., including anti-anti-Id antibodies), small molecule immunopharmaceuticals ("SMIPs™"), single chain or tandem diabodies (TandAbs®), VHHs, Anticalin®, Nanobody® minibodies, BiTEs®, ankyrin repeat proteins or DARPIN®, Avimers®, DARTs, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MicroProteins, Fynomer®, Centyrin®, KALBITOR®, CARs, engineered TCRs, and antigen-binding fragments of any of the above.

[0023] In various embodiments, an antibody comprises one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR) or a variable domain. In some embodiments, an antibody may be a covalently modified ("conjugated") antibody (e.g., an antibody comprising a polypeptide comprising one or more canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular antigen, where the polypeptide is covalently linked to one or more of a therapeutic agent, a detectable moiety, another polypeptide, a carbohydrate moiety, or a polyethylene glycol molecule). In some embodiments, the sequence elements of the antibody are humanized, primatized, chimerized, etc., as known in the art.

[0024] An antibody comprising a heavy chain constant domain can be of any known class of antibody, including, but not limited to, IgA, secretory IgA, IgG, IgE, and IgM, based on the heavy chain constant domain amino acid sequence (e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ)). IgG subclasses are also well known to those of skill in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the class or subclass of antibody (e.g., IgM or IgG1) that is encoded by the heavy chain constant region gene. As used herein, a "light chain" can be of a distinct type (e.g., kappa (κ) or lambda (λ)) based on the amino acid sequence of the light chain constant domain. In some embodiments, an antibody has one or more constant region sequences characteristic of mouse, rabbit, primate, or human immunoglobulins. Naturally produced immunoglobulins are typically glycosylated on the CH2 domain. As is known in the art, the affinity and / or other binding attributes of the Fc region for an Fc receptor can be modulated through glycosylation or other modifications. In some embodiments, an antibody may lack covalent modifications (e.g., glycan attachments) that it would have if produced naturally. In some embodiments, an antibody produced and / or utilized in accordance with the present invention comprises a glycosylated Fc domain (including an Fc domain with such glycosylation that has been modified or engineered).

[0025] Antibody fragment: As used herein, "antibody fragment" refers to a portion of an antibody or antibody drug described herein, typically an antigen-binding portion or a portion that includes a variable region thereof. Antibody fragments can be generated by any means. For example, in some embodiments, antibody fragments can be generated enzymatically or chemically by fragmentation of an intact antibody or antibody drug. Alternatively, in some embodiments, antibody fragments can be generated recombinantly (i.e., by expression of an engineered nucleic acid sequence). In some embodiments, antibody fragments can be wholly or partially synthetically generated. In some embodiments, antibody fragments (particularly antigen-binding antibody fragments) can have a length of at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 amino acids or more, in some embodiments at least about 200 amino acids.

[0026] Related to: As used herein, two events or entities are "related" to each other when the presence, level, and / or form of one event or entity correlates with the other. For example, a particular entity (e.g., a polypeptide, gene signature, metabolite, microorganism, etc.) is considered to be related to a particular disease, disorder, or condition when its presence, level, and / or form correlates with the occurrence and / or susceptibility of the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "related" to each other when these entities directly or indirectly interact with each other to keep them in physical proximity to each other. In some embodiments, two or more entities that are physically related to each other are covalently bound to each other, and in some embodiments, two or more entities that are physically related to each other are not covalently bound to each other, but are non-covalently associated, for example, by hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0027] Between or From: As used herein, the term "between" refers to content that falls between (inclusive of) a stated upper and lower, or first and second boundary (or "boundary") boundary. Similarly, the term "from," when used in the context of a range of values, indicates that the range includes content that lies between (inclusive of) a stated upper and lower limit, or first and second boundary.

[0028] Bioavailability: As used herein, the term "bioavailability" can refer to the extent to which a substance administered to an in vivo subject (e.g., a polypeptide such as an antibody or antibody fragment) becomes available in the tissues targeted by the substance (e.g., bloodstream and / or plasma). Bioavailability can refer to the extent to which a substance administered to an in vivo subject is delivered to the blood of the subject. Bioavailability can refer to the ability of a substance to perform a function in a subject. Bioavailability can be measured in several ways (e.g., as the concentration of a substance in the bloodstream or plasma). In some embodiments, bioavailability can be assessed, for example, by comparing the "area under the curve" (AUC) in a plot of plasma concentration as a function of time (area under the plasma concentration curve from time zero to the time the plasma concentration returns to baseline levels). AUC can be calculated, for example, using the linear trapezoidal rule. "AUC0-t" refers to the area under the plasma concentration curve from time zero to time t, e.g., to the time baseline is reached.

[0029] Cancer: As used herein, the term "cancer" refers to a disease, disorder, or condition in which cells exhibit relatively abnormal, uncontrolled, and / or autonomous proliferation, resulting in an abnormally elevated proliferation rate and / or an abnormal growth phenotype characterized by a marked loss of control over cell proliferation. In some embodiments, cancer may include one or more tumors. In some embodiments, cancer may be or include cells that are pre-cancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. In some embodiments, cancer may be or include a solid tumor. In some embodiments, cancer may be or include a hematological tumor.

[0030] Engineered: As used herein, the term "engineered" refers to the aspect of having been manipulated by the hand of man. For example, a polynucleotide is considered to be "engineered" when two or more sequences that are not naturally linked in that order are manipulated by the hand of man to be joined together in the engineered polynucleotide. One of skill in the art will appreciate that an "engineered" nucleic acid or amino acid sequence can be a recombinant nucleic acid or amino acid sequence. In some embodiments, an engineered polynucleotide includes coding and / or regulatory sequences found in nature operably linked to a first sequence, but not found in nature operably linked to a second sequence, that are present in the engineered polynucleotide and operably linked to the second sequence by the hand of man. In some embodiments, a cell or organism is considered to be "engineered" when it has been treated such that its genetic information has been altered (e.g., new genetic material not previously present has been introduced, e.g., by transformation, mating, somatic hybridization, transfection, transduction, or other mechanisms, or previously present genetic material has been altered or removed, e.g., by substitution, deletion, or mating). This is common practice, and one of skill in the art will understand that complete or incomplete progeny or copies of an engineered polynucleotide or cell are typically still referred to as "engineered," even if the direct manipulation is of a prior entity.

[0031] Excipient: As used herein, "excipient" refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example, to provide or contribute to a desired viscosity or stabilizing effect. In some embodiments, suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like.

[0032] "Improve," "Increase," "Inhibit," or "Reduce": As used herein, "improve," "increase," "inhibit," and "reduce," and their grammatical equivalents, indicate a qualitative or quantitative difference from a baseline.

[0033] Pharmaceutically acceptable: As used herein, the term "pharmaceutical acceptable" as applied to one or more or all of the component(s) for the formulation of the compositions disclosed herein means that each component must be compatible with the other components of the composition and not deleterious to the recipient thereof.

[0034] Pharmaceutical composition or formulation: As used herein, the term "pharmaceutical composition," or "formulation," refers to a composition in which a therapeutic agent is formulated with one or more pharma- ceutical acceptable carriers.

[0035] Polypeptide: As used herein, "polypeptide" refers to a polymeric chain of two or more amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced by the act of man. In some embodiments, a polypeptide may be or include natural amino acids, unnatural amino acids, or both. In some embodiments, a polypeptide may be or include only natural amino acids or only unnatural amino acids. In some embodiments, a polypeptide may include D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may include only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications (e.g., one or more amino acid side chains), for example, at the N-terminus of the polypeptide, the C-terminus of the polypeptide, non-terminal amino acids, or any combination thereof. In some embodiments, such pendant groups or modifications can be selected from acetylation, amidation, lipidation, methylation, phosphorylation, glycosylation, glycation, sulfation, mannosylation, nitrosylation, acylation, palmitoylation, prenylation, pegylation, etc. (including combinations thereof). In some embodiments, the polypeptide can be cyclic and / or can include a cyclic moiety.

[0036] In some embodiments, the term "polypeptide" may be appended to the name of a reference polypeptide, activity, or structure to denote a class of polypeptides that share a related activity or structure. For such classes, the specification provides exemplary polypeptides within the class whose amino acid sequences and / or functions are known and / or which one of skill in the art would recognize. In some embodiments, members of a polypeptide class or family exhibit significant sequence homology or identity to reference polypeptides of the class, share common sequence motifs (e.g., characteristic sequence elements), and / or share a common activity (in some embodiments, activity at a similar level or within a specified range) For example, in some embodiments, a member polypeptide exhibits a degree of overall sequence homology with a reference polypeptide of at least about 30-40%, often about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, and / or contains at least one region (e.g., a conserved region which in some embodiments may be or contain a distinctive sequence element) that exhibits very high sequence identity, often greater than 90%, or even greater than 95%, 96%, 97%, 98%, or 99%. Such a conserved region typically encompasses at least 3-4, and sometimes up to 20 or more amino acids, and in some embodiments the conserved region encompasses at least a stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more contiguous amino acids. In some embodiments, the related polypeptide may be or may comprise a fragment of a parent polypeptide.In some embodiments, a useful polypeptide may be or may comprise multiple fragments, each of which is found in a different spatial arrangement from one another within the same parent polypeptide than is found within the polypeptide of interest, such that the polypeptide of interest is a derivative of that parent polypeptide (e.g., fragments that are directly linked within the parent may be spatially separated within the polypeptide of interest or vice versa, and / or the fragments may be present in a different order within the polypeptide of interest than the parent).

[0037] Reference: As used herein, "reference" refers to a standard or control against which a comparison is made. For example, in some embodiments, an agent, sample, sequence, subject, animal, or individual, or a population thereof, or a measurement or characteristic representative thereof, is compared to a reference, agent, sample, sequence, subject, animal, or individual, or a population thereof, or a measurement or characteristic representative thereof. In some embodiments, the reference is a measurement. In some embodiments, the reference is an established standard or expected value. In some embodiments, the reference is a historical standard. The reference may be quantitative or qualitative. Typically, as will be understood by those of skill in the art, the reference and the values ​​to which the reference is compared correspond to measurements under comparable conditions. Those of skill in the art will understand when there is sufficient similarity to justify reliance and / or comparison. In some embodiments, a suitable reference may be an agent, sample, sequence, subject, animal, or individual, or a population thereof, under conditions that will be recognized as comparable by those of skill in the art, for example, for the purpose of evaluating one or more particular variables (e.g., the presence or absence of an agent or condition), or a measurement or characteristic representative thereof.

[0038] Small molecule: As used herein, the term "small molecule" refers to organic and / or inorganic compounds of low molecular weight. Generally, a "small molecule" refers to a molecule of size less than about 5 kilodaltons (kD). In some embodiments, a small molecule is less than about 4 kD, 3 kD, 2 kD, or 1 kD. In some embodiments, a small molecule is less than about 800 Daltons (D), less than about 600 Daltons, less than about 500 Daltons, less than about 400 Daltons, less than about 300 Daltons, less than about 200 Daltons, or less than about 100 Daltons. In some embodiments, a small molecule is less than about 2000 g / mol, less than about 1500 g / mol, less than about 1000 g / mol, less than about 800 g / mol, or less than about 500 g / mol. In some embodiments, a small molecule is not a polymer. In some embodiments, a small molecule does not include a polymeric portion. In some embodiments, a small molecule is not a polypeptide and / or does not include a polypeptide. In some embodiments, a small molecule is not and / or does not comprise a polynucleotide (e.g., is not an oligonucleotide). In some embodiments, a small molecule is not and / or does not comprise a polysaccharide (e.g., in some embodiments, a small molecule is not a glycoprotein, proteoglycan, glycolipid, etc.). In some embodiments, a small molecule is not a lipid. In some embodiments, a small molecule is a modulator (e.g., is an inhibitor or activator). In some embodiments, a small molecule is biologically active. In some embodiments, a small molecule is detectable (e.g., comprises at least one detectable moiety). In some embodiments, a small molecule is a therapeutic agent.

[0039] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, a rat, or a mouse). In some embodiments, the subject suffers from a disease, disorder, or condition. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject does not suffer from a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject has one or more characteristics characteristic of a susceptibility or risk for a disease, disorder, or condition. In some embodiments, the subject is a subject who has been tested for a disease, disorder, or condition and / or to whom a treatment has been administered. In some cases, a human subject may be referred to interchangeably as a "patient" or an "individual." A subject who has been administered an agent relevant to the treatment of a disease, disorder, or condition to which the subject is associated may be referred to as a subject in need of the agent, i.e., a subject in need thereof.

[0040] Therapeutic Agent: As used herein, the term "therapeutic agent" refers to any agent that elicits a desired pharmacological effect when administered to a subject. In some embodiments, an agent is considered a therapeutic agent if it exhibits a statistically significant effect in the entire appropriate population. In some embodiments, the appropriate population may be a population of model organisms or a human population. In some embodiments, the appropriate population may be defined by various criteria, such as a certain age group, sex, genetic background, pre-existing clinical conditions, etc. In some embodiments, a therapeutic agent is a substance that can be used to treat a disease, disorder, or condition. In some embodiments, a "therapeutic agent" is a drug that has been approved, or requires approval, by a government agency before it can be marketed for administration to humans. In some embodiments, a "therapeutic agent" is a drug that requires a medical prescription for administration to humans.

[0041] Therapeutically effective amount: As used herein, "therapeutically effective amount" refers to an amount that produces a desired effect upon administration. In some embodiments, the term refers to an amount that, when administered to a population suffering from or susceptible to a disease, disorder, and / or condition, is sufficient to treat the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is an amount that reduces the occurrence and / or severity of, and / or delays the onset of, one or more symptoms of a disease, disorder, and / or condition. Those skilled in the art will appreciate that a therapeutically effective amount will not necessarily achieve successful treatment in all individuals undergoing a particular treatment. Rather, a therapeutically effective amount may be an amount that, when administered to a patient in need of such treatment, produces a particular desired pharmacological response in a meaningful number of subjects. In some embodiments, reference to a therapeutically effective amount may be a reference to an amount as measured in one or more specific tissues (e.g., tissues affected by a disease, disorder, or condition) or bodily fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). One of skill in the art will appreciate that in some embodiments, a therapeutically effective amount of a particular agent or therapy may be formulated and / or administered in a single dose, hi some embodiments, a therapeutically effective agent may be formulated and / or administered in multiple doses, e.g., as part of a dosing regimen.

[0042] Treatment: As used herein, the term "treatment" (and "treat" or "treating") refers to the administration of a therapy administered with the intent to partially or completely alleviate, ameliorate, mitigate, inhibit, delay onset, reduce severity, and / or reduce occurrence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, or condition, or to achieve any such result. In some embodiments, such treatment may be of a subject who does not exhibit symptoms of the associated disease, disorder, and / or condition, and / or a subject who exhibits only early symptoms of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established symptoms of the associated disease, disorder, and / or condition. In some embodiments, the treatment may be of a subject who has been diagnosed as suffering from the associated disease, disorder, and / or condition. In some embodiments, the treatment may be of a subject who is known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the associated disease, disorder, or condition. [Brief description of the drawings]

[0043] [Figure 1] 1 is a set of four images showing trastuzumab microparticles before (left panel) and after drying (right panel).

[0044] [Diagram 2] 1 is an image showing microparticles of liraglutide produced by a typical crystallization approach known to one of skill in the art.

[0045] [Diagram 3] 1 is an image showing microparticles of liraglutide produced by a typical crystallization approach known to one of skill in the art.

[0046] [Figure 4] 1 is an image showing microparticles of liraglutide produced by a typical crystallization approach known to one of skill in the art.

[0047] [Diagram 5] 1 is an image showing microparticles of liraglutide produced by a typical crystallization approach known to one of skill in the art.

[0048] [Figure 6] 1 is an image showing microparticles of liraglutide produced by a typical crystallization approach known to one of skill in the art.

[0049] [Figure 7] 1 is an image showing microparticles of liraglutide produced by a typical crystallization approach known to one of skill in the art.

[0050] [Figure 8] 1 is an image showing microparticles of liraglutide produced by a typical crystallization approach known to one of skill in the art.

[0051] [Figure 9] 1 is an image showing microparticles of liraglutide produced by a typical crystallization approach known to one of skill in the art.

[0052] [Figure 10] 1 is an image showing microparticles of insulin produced by a typical crystallization approach known to one of skill in the art.

[0053] [Figure 11] 1 is an image showing microparticles of insulin produced by a typical crystallization approach known to one of skill in the art.

[0054] [Figure 12] 1 is an image showing microparticles of insulin produced by a typical crystallization approach known to one of skill in the art.

[0055] [Figure 13] 1 is an image showing microparticles of insulin produced by a typical crystallization approach known to one of skill in the art.

[0056] [Figure 14] 1 is an image showing microparticles of insulin produced by a typical crystallization approach known to one of skill in the art.

[0057] [Figure 15] 1 is an image showing microparticles of insulin produced by a typical crystallization approach known to one of skill in the art.

[0058] [Figure 16] 1 is an image showing microparticles of insulin produced by a typical crystallization approach known to one of skill in the art.

[0059] [Figure 17] 1 is a graph showing plasma concentrations of liraglutide over time following subcutaneous administration of a standard therapeutic formulation of liraglutide or oral administration of a formulation provided herein.

[0060] [Figure 18] 1 is a pair of graphs showing plasma concentrations of liraglutide over time following subcutaneous administration of a standard therapeutic formulation of liraglutide or oral administration of a formulation provided herein.

[0061] [Figure 19] 1 is a pair of graphs showing plasma concentrations of liraglutide over time following subcutaneous administration of a standard therapeutic formulation of liraglutide or oral administration of a formulation provided herein.

[0062] [Figure 20] 1 is a graph showing plasma concentrations of trastuzumab over time following subcutaneous administration of a standard of care formulation of trastuzumab or oral administration of a formulation provided herein.

[0063] [Figure 21]1 is a graph showing plasma concentrations of trastuzumab over time following subcutaneous administration of a standard of care formulation of trastuzumab or oral administration of a formulation provided herein.

[0064] [Figure 22] 1 is a graph showing plasma concentrations of insulin over time following subcutaneous administration of a standard of care formulation of insulin or oral administration of a formulation provided herein.

[0065] [Diagram 23] FIG. 1 shows plasma concentrations of trastuzumab over time following subcutaneous administration of a standard of care formulation of trastuzumab or oral administration of a formulation containing vitamin E, coconut oil, cod liver oil, or ghee.

[0066] [Figure 24] 1 is a graph showing plasma concentrations of liraglutide following administration of liraglutide oral formulations to New Zealand White rabbits.

[0067] [Diagram 25] 1 is a graph showing plasma concentrations of liraglutide following administration of a liraglutide intravenous formulation to New Zealand White rabbits.

[0068] [Figure 26] 1 is a graph showing plasma concentrations of liraglutide following administration of a subcutaneous liraglutide formulation to New Zealand White rabbits. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0069] The pharmaceutical industry has expended great efforts to bring polypeptide therapeutics to market. Hundreds of polypeptides have been used in clinical trials for the treatment of over 100 conditions. However, clinical use of polypeptides is hampered by many obstacles that impede their successful delivery. Factors considered in the development of formulations for polypeptide delivery include bioavailability, stability, and therapeutic efficacy. Many polypeptides are administered by parenteral routes, such as subcutaneous, intramuscular, and intravenous injections. The present disclosure includes the recognition that oral administration is generally preferable to injection of polypeptide therapeutics, for example, with respect to patient tolerance, home use, and compliance with long-term regimens. Due to these advantages, among others, oral formulations of polypeptide drugs are believed to have different and / or greater therapeutic and commercial value than parenteral formulations.

[0070] Despite the great efforts being made to achieve oral administration of polypeptides, parenteral delivery remains the predominant mode of administration for polypeptide therapeutics. While oral administration may be the standard for the administration of small molecules, the difficulty of oral administration of polypeptides is a problem recognized by those skilled in the art. Factors that make oral administration of polypeptides very difficult include intrinsic physicochemical and biological properties, including large molecular size, poor permeability through gastrointestinal membranes, low stability due to the low pH of gastric juices, and susceptibility to proteolytic enzymes. In various studies, it has been observed that orally administered polypeptides exhibit bioavailability of less than 1%, but higher levels, in some cases target bioavailability of at least 30%-50%, are considered preferable for therapeutic efficacy. Efforts to improve polypeptide stability and performance include chemical modification of the polypeptide (e.g., PEGylation, hyperglycosylation, and mannosylation) or the use of colloidal carriers (including microparticles, nanoparticles, liposomes, carbon nanotubes, and micelles). Despite such efforts, parenteral administration of polypeptides has remained the standard. The present disclosure provides a solution to the long-standing problem of formulating polypeptides for oral administration.

[0071] Various compositions of the present disclosure may comprise a core within a pharma- ceutically acceptable capsule. In various embodiments, the core comprises an amorphous or crystallized polypeptide composition and a pharma- ceutically acceptable carrier comprising a vitamin E agent.

[0072] Vitamin E As used herein, vitamin E agent refers to a compound or entity that delivers vitamin E active moiety to a subject when administered to the subject. In some embodiments, vitamin E agent is provided and / or utilized as a salt, cocrystal, free acid or base, solvate, ester, hydrate, polymorph, or anhydrous form. In some embodiments, vitamin E agent is provided and / or utilized in a specific stereoisomeric form or as a mixture of stereoisomeric forms. In some embodiments, vitamin E agent is a prodrug of vitamin E, and vitamin E is the metabolite that is intended to have a therapeutic effect.

[0073] A vitamin E agent (interchangeably referred to herein as vitamin E) may be or include one or more vitamin E tocols selected from one or more vitamin E tocopherols and / or one or more vitamin E tocotrienols. Natural vitamin E tocols include two series of compounds: tocopherols with saturated side chains and tocotrienols with unsaturated side chains. Tocopherols and tocotrienols have similar chemical structures, characterized by a long isoprenoid side chain attached at the 2-position of a 6-chromanol ring. Tocopherols contain a chromanol ring and a 16-carbon tail. Tocotrienols differ from tocopherols in that they have a farnesyl rather than a saturated isoprenoid C16 side chain. Vitamin E tocols (e.g., tocopherols and tocotrienols) are named as α, β, γ, or δ based on the methylation pattern of the chromanol ring. β- and γ- are dimethylated at the 5- and 8-positions, or the 7- and 8-positions, respectively, of the chromanol ring. α-tocol is trimethylated at the 5-, 7-, and 8-positions of the chromanol ring, and δ- is monomethylated at the 8-position of the chromanol ring. Vitamin E preparations can include α-, β-, γ-, or δ-tocols, or mixtures thereof. For example, vitamin E tocopherols can include any one or more of tocopherol α, β, γ, and δ, as well as derivatives thereof. Vitamin E tocotrienols can include any one or more of tocotrienol α, β, γ, and δ, as well as derivatives thereof. Of these vitamin E alcohols, α-tocopherol and γ-tocopherol are the most abundant in nature. Certain commercially available vitamin E supplements can generally include α-tocopherol. Certain commercially available vitamin E supplements can generally include a mixture of vitamin E tocols having various specific structures.

[0074] The vitamin E agent may include all stereoisomeric (e.g., enantiomeric or diastereomeric) forms and all geometric or conformational isomeric forms of the vitamin E agent. For example, the R and S configurations of each stereocenter of the vitamin E agent are contemplated as part of the present disclosure. Thus, single stereochemical isomers of the vitamin E agent, as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures, are within the scope of the present disclosure. For example, the vitamin E agent may be or include a stereoisomer of vitamin E tocopherol or tocotrienol. The tocopherol molecule has at least three stereocenters at C-2, C-4', and C-8', allowing at least eight stereoisomers. For example, the vitamin E agent may be or include a stereoisomer of α-tocopherol selected from RRR, RRS, RSS, SSS, RSR, SRS, SRR, and SSR. In various embodiments, the RRR stereoisomer is naturally occurring. In some embodiments, vitamin E agents contain a mixture of one or more stereoisomers, such as one or more α-tocopherol stereoisomers (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 stereoisomers). Natural tocopherol exists in the RRR-configuration, whereas synthetic forms can contain eight different stereoisomers, and are referred to as all-rac-alpha-tocopherol. Tocotrienol has only one stereocenter at C-2, and naturally occurring tocotrienol exists exclusively in the 2R,3'E,7'E configuration.

[0075] Vitamin E agents include derivatives and / or analogs of vitamin E tocol.Vitamin E can be modified in various ways, for example, to the chroman moiety of vitamin E tocol.Examples of vitamin E tocol derivatives include 2-, 5-, or 6-substituted chroman derivatives.Vitamin E tocol can be derivatized by conjugation with agents such as PEG moieties (PEGylation) or acids (e.g., hyaluronic acid).

[0076] The vitamin E agent may be or include a prodrug of vitamin E tocol, such as an ester of vitamin E tocol (e.g., acetate, succinate, or nicotinate). In certain embodiments, the ester of the vitamin E agent is prepared from the phenolic form of the vitamin E agent using conventional methods known in the art. Tocopheryl esters (e.g., alpha-tocopheryl acetate, tocopheryl succinate, tocopheryl nicotinate, tocopheryl linoleate, alpha-tocopheryl phosphate, etc.) may exhibit reduced susceptibility to oxidation. Tocopherol esters are de-esterified in the intestine (e.g., by the enzyme esterase) and absorbed as free tocopherol. The vitamin E agent may be or include a non-esterified vitamin E tocol. It is understood that in various embodiments, free and esterified vitamin E tocols have comparable bioavailability.

[0077] The present disclosure includes vitamin E formulations that are absorbed, for example, by the human body. The present disclosure includes both liquid and solid forms of vitamin E. An exemplary solid form of vitamin E can be a white to light tan granular powder. The liquid form of vitamin E can be insoluble in water, soluble in alcohol, and / or miscible (e.g., in ether, acetone, vegetable oil, and / or chloroform). Various liquid forms of vitamin E are clear, yellow to brownish red, and / or viscous oils. The present disclosure includes fat-soluble vitamin E formulations. For example, in various embodiments, a solid form such as alpha tocopheryl succinate is insoluble in water but soluble in fats (e.g., vegetable oils) and can, in various embodiments, coat crystallized polypeptides and / or polypeptide particles.

[0078] The pharma- ceutically acceptable carriers that contain vitamin E agents can include vitamin E agents (e.g., fat-soluble vitamin E agents) and oils. In various embodiments, vitamin E can function as a dietary supplement in addition to the carrier. In various embodiments, the pharma-ceutically acceptable carriers that contain vitamin E agents (e.g., oils that contain naturally occurring or added vitamin E agents) can include vitamin E agents and oils that are plant-derived oils, and optionally the plant-derived oils are selected from wheat germ oil, hazelnut oil, canola / rapeseed oil, sunflower oil, safflower oil, almond oil, grapeseed oil, sunflower kernel, almond, almond butter, wheat germ, canola oil, palm oil, peanut oil, margarine, tab, hazelnut, corn oil, olive oil, soybean oil, pine nuts, peanut butter, and peanuts. Exemplary concentrations of vitamin E in certain such compositions are shown in Table 1. While these concentrations shown are merely exemplary and, in some cases, reflect typical or naturally occurring amounts of vitamin E agents, the present disclosure includes that the amount of vitamin E agent in the oil composition may be altered, for example, by adding vitamin E to achieve the amounts or concentrations of vitamin E disclosed herein for use in suspension or encapsulation formulations of crystallized or amorphous polypeptides. [Table 1]

[0079] Pharmaceutically Acceptable Carriers The formulation of the present disclosure may include a pharma- ceutically acceptable carrier that is or contains a vitamin E agent.As used herein, the term "pharma-ceutically acceptable carrier" refers to a pharma-ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that facilitates the formulation of a drug (e.g., a pharmaceutical drug), modifies the bioavailability of the drug, or facilitates the transport of the drug from one organ or part of a subject to another. Some examples of materials that can function as pharma- ceutically acceptable carriers include sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients; oils, such as peanut oil, cottonseed oil, virgin coconut oil, almond oil, malt oil, any edible oil, safflower oil, sesame oil, olive oil, corn oil, and the like. Examples of suitable pharmaceutical excipients include: corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffers; polyester, polycarbonate, and / or polyanhydrides; and other non-toxic compatible substances used in pharmaceutical formulations. Excipients can include non-therapeutic agents that may be included in the pharmaceutical composition, for example, to provide or contribute to a desired viscosity or stabilizing effect. In some embodiments, suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, cocoa butter, suppository wax, glycerol, propylene glycol, water, ethanol, etc.In various embodiments, the polypeptide formulations of the present disclosure (e.g., encapsulation cores of polypeptide formulations or suspensions) further comprise one or more pharma- ceutically acceptable carriers. In various specific embodiments, the one or more pharma-ceutically acceptable carriers are selected from the group consisting of aggregation reducing agents, sugars or sugar alcohols, polysaccharides, stabilizers, hyaluronidase, buffers, preservatives, carriers, antioxidants, chelating agents, natural or synthetic polymers, cryoprotectants, lyoprotectants, surfactants, bulking agents, acidifying agents, components that reduce injection site discomfort, antifoaming agents, chelating agents, natural or synthetic polymers, cryoprotectants, cryoprotectants, surfactants, bulking agents, acidifying agents, components that reduce injection site discomfort, antifoaming agents, alkalizing agents, vehicles, aggregation inhibitors, solubilizers, tonicity adjusting agents, and stabilizers, and combinations thereof.

[0080] In various embodiments, the aggregation reducing agent is selected from the group consisting of nicotinic acid, caffeine citrate, caffeine nicotinate, caffeine, octyl-β-D-glucopyranoside, and n-dodecyl-β-D-maltoside, optionally in combination with arginine, tryptophan, histidine, proline, cysteine, methionine, β-alanine, potassium glutamate, arginine ethyl ester, lysine, aspartic acid, glutamic acid, glycine, DTPA (diethylenetriaminepentaacetic acid), EGTA (aminopolycarboxylic acid), EDTA (ethylenediaminetetraacetic acid), hydroxypropyl beta (H P-beta) cyclodextrin, hydroxypropyl gamma (HP-gamma) cyclodextrin, sulfobutyl ether (SBE) cyclodextrin, TMAO (trimethylamine N-oxide) trehalose, ethylene glycol, betaine, xylitol, sorbitol, 6-(N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid (NBD-X), methyl acetyl phosphate (MAP), citraconic anhydride, pyrophosphate, citrate, and combinations with one or more of any of the foregoing combinations.

[0081] In various embodiments, the tonicity adjusting agent may include one or more of arginine, cysteine, histidine, glycine, sodium chloride, potassium chloride, sodium citrate, a sugar such as sucrose, glucose, dextrose, glycerin, or mannitol, and combinations thereof.

[0082] In various embodiments, the antioxidant may include one or more of glycine, lysine, EDTA, DTPA, sorbitol, mannitol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, sodium thiosulfate, sulfur dioxide, tocopherol, and combinations thereof.

[0083] In various embodiments, the lyoprotectant may include one or more of sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, maltose, lactulose, maltulose, glucitol, maltitol, lactitol, isomaltulose, and mannitol; amino acids (e.g., arginine, histidine, proline, or glycine); lyotropic salts (e.g., magnesium sulfate); propylene glycol, glycerol, poly(ethylene glycol), or poly(propylene glycol); gelatin, dextrin, modified starch, carboxymethylcellulose, and combinations thereof.

[0084] In various embodiments, a pharma- ceutically acceptable carrier explicitly excludes pharma- ceutically active agents. In various embodiments, a pharma- ceutically acceptable carrier explicitly excludes one or more, or all, polypeptides.

[0085] In various embodiments in which a polypeptide formulation for oral delivery comprises a core or suspension comprising an amorphous or crystallized polypeptide composition and a pharma- ceutically acceptable carrier comprising a vitamin E agent, the core, suspension, or pharma- ceutically acceptable carrier may be characterized by an amount of vitamin E agent that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% by weight, molar ratio, or volume. In various embodiments, the core, suspension, or pharma- ceutically acceptable carrier may be characterized by an amount of vitamin E agent that is between 10%-90%, between 20%-90%, between 30%-90%, between 40%-90%, between 50%-90%, between 60%-90%, between 70%-90%, between 80%-90%, between 10%-70%, between 20%-70%, between 30%-70%, between 40%-70%, between 50%-70%, between 60%-70%, between 10%-50%, between 20%-50%, between 30%-50%, or between 40%-50% on a weight, molar, or volume basis.

[0086] Crystallized and amorphous polypeptides The present disclosure includes oral formulations comprising vitamin E and a polypeptide (e.g., a polypeptide present in an amorphous polypeptide composition or a crystallized polypeptide composition). Pharmaceutical agents may exist in various forms, including polymorphs, solvates, hydrates, salts, co-crystals, and amorphous solids.

[0087] Amorphous polypeptide compositions may include compositions in which the polypeptide molecules are disordered or essentially disordered. Amorphous polypeptides may lack or essentially lack long-range order in the positions of atoms. In various embodiments, amorphous polypeptides may be more soluble than crystallized forms of the same polypeptide. In certain embodiments, amorphous polypeptides are compositions that have not been crystallized and / or processed according to a method of crystallization. In certain embodiments, amorphous polypeptide compositions may include regions of short-range order, residual crystallinity, polymorphic states, and different densities, none of which necessarily constitute long-range order. In various embodiments, amorphous polypeptide compositions may include a portion of a crystallized polypeptide, e.g., a portion of a crystallized polypeptide that is less than 20% of the total polypeptide by mass, volume, or mole (e.g., less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the crystallized polypeptide). Techniques for quantifying crystallinity include XRD, DSC, solution calorimetry, moisture sorption, isothermal calorimetry, and thermally stimulated current (TSC). In various embodiments, the amorphous polypeptide composition does not diffract X-rays in a coherent manner and / or the powder X-ray diffraction pattern is a broad halo with no or very few characteristic peaks.

[0088] In various embodiments, amorphous polypeptide compositions of the disclosure comprise an average and / or maximum particle size of less than 25 microns, e.g., less than 20, 15, 10, 5, 4, 3, 2, 1, or 0.5 microns, e.g., optionally the particles have an average and / or maximum particle size of between 0.5 microns and 1, 2, 3, 4, 5, 10, 15, 20, or 25 microns, or the particles of polypeptide have an average and / or maximum particle size of between 1 micron and 1, 2, 3, 4, 5, 10, 15, 20, or 25 microns. In various embodiments, the particles of the polypeptide compositions of the disclosure are microparticles of polypeptide.

[0089] Amorphous polypeptide compositions of the disclosure include compositions that contain a high percentage of amorphous and / or non-crystallized polypeptides (e.g., of polypeptides characterized by a particular amino acid sequence) relative to other agents or types of agents. In various embodiments, amorphous polypeptide compositions of the disclosure contain at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amorphous and / or non-crystallized polypeptides (e.g., of polypeptides characterized by a particular amino acid sequence) based on the weight, molar ratio, or volume of the composition or polypeptides present in the composition. In various embodiments, an amorphous polypeptide composition may be characterized by an amount of amorphous and / or non-crystallized polypeptide (e.g., of a polypeptide characterized by a particular amino acid sequence) that is between a lower limit of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, or 90%, based on the weight, molar ratio, or volume of the composition or polypeptide present in the composition, to an upper limit of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, based on the weight, molar ratio, or volume of the composition or polypeptide present in the composition.

[0090] In various embodiments, the amorphous polypeptide compositions of the disclosure are free or substantially free of non-polypeptide drugs (and / or drugs other than amorphous polypeptides), e.g., the amorphous polypeptide compositions comprise no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% non-polypeptide drugs (and / or drugs other than amorphous polypeptides) by weight, molar ratio, or volume. In various embodiments, the amorphous polypeptide compositions of the disclosure comprise no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% non-polypeptide drugs (and / or drugs other than amorphous polypeptides) by weight, molar ratio, or volume. In various embodiments, an amorphous polypeptide composition comprises particles of one or more particular polypeptides and is free or substantially free of other polypeptide agents (optionally including crystallized form(s) of one or more particular polypeptides), e.g., an amorphous polypeptide composition comprises no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% of other polypeptide agents by weight, molar ratio, or volume. In various embodiments, an amorphous polypeptide composition comprises no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of other polypeptide agents by weight, molar ratio, or volume. In various embodiments, an amorphous polypeptide composition comprises particles of one or more particular polypeptides and is free or substantially free of other agents (optionally including crystallized form(s) of one or more particular polypeptides), e.g., an amorphous polypeptide composition comprises no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% of other agents by weight, molar ratio, or volume. In various embodiments, an amorphous polypeptide composition comprises no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of other agents by weight, molar ratio, or volume.

[0091] In various embodiments, amorphous polypeptide compositions of the present disclosure comprise particles of one or more specific polypeptides, which particles of the one or more specific polypeptides are free or substantially free of non-polypeptide agents, e.g., the particles comprise no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% non-polypeptide agents by weight, molar ratio, or volume. In various embodiments, amorphous polypeptide compositions of the present disclosure comprise particles of one or more specific polypeptides, which particles of the one or more specific polypeptides comprise no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% non-polypeptide agents by weight, molar ratio, or volume. In various embodiments, amorphous polypeptide compositions of the present disclosure comprise particles of one or more specific polypeptides that are free or substantially free of other polypeptides, e.g., particles of one or more specific polypeptides that comprise no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% other polypeptides by weight, molar ratio, or volume. In various embodiments, amorphous polypeptide compositions of the present disclosure comprise particles of one or more specific polypeptides that comprise no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% other polypeptides by weight, molar ratio, or volume. In various embodiments, amorphous polypeptide compositions of the present disclosure comprise particles of one or more particular polypeptides that are free or substantially free of other agents (optionally including non-crystallized form(s) of the one or more particular polypeptides), e.g., particles of the one or more particular polypeptides that contain no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% of other agents by weight, molar ratio, or volume.In various embodiments, amorphous polypeptide compositions of the present disclosure comprise particles of one or more particular polypeptides that contain up to 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% other agents by weight, molar ratio, or volume.

[0092] Processes for producing amorphous polypeptide compositions can include molecular quenching of melts, rapid precipitation by addition of antisolvents, freeze-drying, spray-drying, spray-freeze-drying, precipitation in supercritical fluids, solid dispersion, and solid-state chemical reaction (decomposition) of crystalline precursors.For example, freeze-drying of protein / PEG blend solutions followed by removal of PEG from the matrix has been found to result in precipitated protein particles in amorphous form.Processes that introduce mechanical or chemical stress (grinding, milling, and wet granulation) can completely or partially amorphize crystalline materials.

[0093] In some embodiments, the amorphous polypeptide composition may be in a hydrated form or may be prepared from a hydrated form. The hydrated form may include an alcohol (e.g., ethanol). In some embodiments, the amorphous polypeptide composition is in a solvated form or may be prepared from a solvated form. Exemplary solvents may include, for example, acidic solvents or organic solvents. In some embodiments, the solvent may include DMSO, DMF, acetic acid, acetonitrile, methanol, propanol, isopropanol, acetone, anisole, 1-butanol, 2-butanol, butyl acetate, ferric / -butyl methyl ether, cumene, dimethyl sulfoxide, ethanol, ethyl acetate, ethyl ether, ethyl formate, formic acid, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, 3-methyl-1-butanol, methyl ethyl ketone, methyl isobutyl ketone, 2-methyl-1-propanol, pentane, 1-pentanol, 1-propanol, 2-propanol, propyl acetate, tetrahydrofuran, ethanol, and / or water. Certain methods of preparing amorphous polypeptide compositions can include removal of solvent from the solvated form by rapid solvent evaporation, spray drying, roller drying, solvent precipitation, or freeze drying. In some embodiments, the amorphous polypeptide composition is free of cations, e.g., 2+ charged cations (e.g., Ba 2+ , Ca 2+ , Cr 2+ , Co 2+ , Cu 2+ , Fe 2+ , Pb 2+ , Mg 2+ , Mn 2+ , Ni 2+ , Sr 2+ , Sn 2+ , or Zn 2+ ).

[0094] Crystal formation may include building an amorphous solid drug into a crystalline solid form. Crystal formation may utilize various intermolecular interactions, including, for example, hydrogen bonds, p-stacking, and van der Waals forces. Hydrogen bond formation is often involved in intermolecular interactions in molecular solids. Typically, crystallization is considered for small molecule drugs, and less for polypeptides. Typically, polypeptides lack a well-defined conformation in solution. In certain embodiments, the amorphous or crystallized polypeptide composition is a powder form that contains crystals of the polypeptide.

[0095] In various embodiments, the crystallized polypeptide compositions of the present disclosure comprise polypeptide crystals having an average and / or maximum particle size of less than 25 microns, e.g., less than 20, 15, 10, 5, 4, 3, 2, 1, or 0.5 microns, e.g., optionally, the polypeptide crystals have an average and / or maximum particle size of between 0.5 microns and 1, 2, 3, 4, 5, 10, 15, 20, or 25 microns, or the polypeptide crystals have an average and / or maximum particle size of between 1 micron and 1, 2, 3, 4, 5, 10, 15, 20, or 25 microns. In various embodiments, the crystals of the crystallized polypeptide compositions of the present disclosure are polypeptide microcrystals.

[0096] Crystallized polypeptide compositions of the present disclosure include compositions that contain a high percentage of crystallized polypeptides (e.g., of polypeptides characterized by a particular amino acid sequence) relative to other agents or types of agents. In various embodiments, crystallized polypeptide compositions of the present disclosure contain at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% crystallized polypeptides (e.g., of polypeptides characterized by a particular amino acid sequence) based on the weight, molar ratio, or volume of the composition or polypeptides present in the composition. In various embodiments, a crystallized polypeptide composition may be characterized by an amount of crystallized polypeptide (e.g., of a polypeptide characterized by a particular amino acid sequence) that is between a lower limit of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, or 90%, based on the weight, molar ratio, or volume of the composition or polypeptide present in the composition, to an upper limit of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, based on the weight, molar ratio, or volume of the composition or polypeptide present in the composition.

[0097] In various embodiments, the crystallized polypeptide compositions of the disclosure are free or substantially free of non-polypeptide agents, e.g., the crystallized polypeptide compositions contain no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% non-polypeptide agents by weight, molar ratio, or volume. In various embodiments, the crystallized polypeptide compositions of the disclosure contain no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% non-polypeptide agents by weight, molar ratio, or volume. In various embodiments, a crystallized polypeptide composition comprises crystals of one or more particular polypeptides and is free or substantially free of other polypeptide agents (optionally including non-crystallized form(s) of one or more particular polypeptides), e.g., a crystallized polypeptide composition comprises no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% of other polypeptide agents by weight, molar ratio, or volume. In various embodiments, a crystallized polypeptide composition comprises crystals of one or more particular polypeptides and comprises no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of other polypeptide agents by weight, molar ratio, or volume. In various embodiments, a crystallized polypeptide composition comprises crystals of one or more particular polypeptides and is free or substantially free of other agents (optionally including non-crystallized form(s) of one or more particular polypeptides), e.g., a crystallized polypeptide composition comprises no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% of other agents by weight, molar ratio, or volume. In various embodiments, a crystallized polypeptide composition comprises crystals of one or more particular polypeptides and comprises no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of other agents by weight, molar ratio, or volume.

[0098] In various embodiments, the crystallized polypeptide compositions of the present disclosure comprise crystals of one or more specific polypeptides, which crystals of the one or more specific polypeptides are free or substantially free of non-polypeptide agents, e.g., the crystals comprise no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% non-polypeptide agents by weight, molar ratio, or volume. In various embodiments, the crystallized polypeptide compositions of the present disclosure comprise crystals of one or more specific polypeptides, which crystals comprise no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% non-polypeptide agents by weight, molar ratio, or volume. In various embodiments, the crystallized polypeptide compositions of the present disclosure comprise crystals of one or more specific polypeptides that are free or substantially free of other polypeptides, e.g., the crystals of the one or more specific polypeptides comprise no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% other polypeptides by weight, molar ratio, or volume. In various embodiments, the crystallized polypeptide compositions of the present disclosure comprise crystals of one or more specific polypeptides that comprise no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% other polypeptides by weight, molar ratio, or volume. In various embodiments, the crystallized polypeptide compositions of the present disclosure comprise crystals of one or more particular polypeptides that are free or substantially free of other agents (optionally including non-crystallized form(s) of the one or more particular polypeptides), e.g., the crystals of the one or more particular polypeptides contain no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% of other agents by weight, molar ratio, or volume.In various embodiments, the crystallized polypeptide compositions of the present disclosure comprise crystals of one or more particular polypeptides that contain up to 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% other agents by weight, molar ratio, or volume.

[0099] Polypeptide crystals can be prepared from a polypeptide sample. Polypeptide crystals can be prepared from a polypeptide sample that is substantially pure from other agents and / or contaminants. Polypeptide crystals can be prepared from amorphous material, e.g., lyophilized material that is amorphous or precipitated amorphous solids. In some embodiments, polypeptide crystals can be prepared from a mixture of amorphous and crystalline material, e.g., lyophilized material that is a mixture of amorphous and crystalline material, or a mixture of precipitated amorphous and crystalline solids. In various embodiments, polypeptide crystals can be prepared from a polypeptide sample having a polypeptide concentration between 0.1 and 200 mg / ml, e.g., a polypeptide sample having a polypeptide concentration between a lower limit of 0.1, 1, 5, 10, 15, 20, 25, 50, 75, or 100 mg / ml and an upper limit of 15, 20, 25, 50, 75, 100, 125, 150, 175, or 200 mg / ml.

[0100] Means of polypeptide crystallization include, but are not limited to, evaporation, slow diffusion (e.g., vapor diffusion at ambient or low temperature), slow cooling, slurrying, hanging drop, sitting drop, seeded crystallization, and / or other crystallization methods known in the art. Those skilled in the art will appreciate that many crystallization methods are well understood and known in the art, and that polypeptides are generally easy to crystallize. Those skilled in the art will further appreciate that polypeptides are particularly easy to crystallize when no particular crystal size is required, when large crystals are not required, when no particular crystal morphology is required, and / or when perfectly regular crystals are not required, any or all of which may characterize various embodiments of the present disclosure.

[0101] Crystalline polypeptides can be prepared by mixing polypeptides in a suitable solvent (e.g., water) and then returning the polypeptide to solid phase. For example, polypeptides can form crystals when precipitated from an aqueous solution (e.g., ammonium sulfate). In certain exemplary embodiments, a polypeptide saturated solution is prepared by increasing the concentration of the polypeptide in the solution. When solubility is maximized, precipitation of the polypeptide can occur, and the precipitant can become crystals. A slow precipitation rate produces a small number of relatively large crystals, while a fast precipitation rate produces a large number of small crystals, so the rate of precipitation is generally not critical to the formation of crystals.

[0102] Evaporation (e.g., slow evaporation) is a common means of crystallizing polypeptides. Precipitation of a polypeptide can occur by evaporating (e.g., slowly evaporating) the solvent of a solution of the polypeptide until the solution reaches saturation, thereby causing precipitation of the polypeptide.

[0103] Cooling (e.g., slow cooling) is another method of crystallizing a polypeptide. Precipitation of a polypeptide can occur by cooling (e.g., slowly cooling) a solution of the polypeptide, thereby reducing the maximum solubility of the polypeptide in the solution and inducing precipitation to occur.

[0104] Vapor diffusion and batch methods are also commonly used for polypeptide crystallization. In vapor diffusion, a droplet containing a mixture of precipitant and unprecipitated polypeptide is sealed in a chamber containing pure precipitant. Water vapor then diffuses from the droplet until the osmolality of the droplet and the precipitate are equal. Dehydration of the droplet slowly concentrates the polypeptide and precipitate until equilibrium can be achieved, favoring crystallization. Vapor diffusion can be performed in either hanging or sitting drop format. In the hanging drop method, a droplet of the polypeptide solution is placed on an inverted coverslip, which is then suspended above a reservoir. In the sitting drop method, the droplet can be placed on a platform separated from the reservoir. Both of these methods require a sealed environment to allow equilibrium to occur between the droplet and the reservoir.

[0105] Batch methods rely on bringing the polypeptide directly to the nucleation zone by mixing it with an appropriate amount of precipitant. Various examples provided herein include batch crystallization. Batch crystallization differs from continuous crystallization in that the crystal product for a batch system is drawn off only once at the end of the batch run. Batch crystallization can also include semi-batch systems, in which one or more feed solutions are added to the crystallizer at a constant or variable rate throughout all or part of the batch. In various embodiments, batch crystallization can vary in volume, for example, from a microliter (e.g., in an Eppendorf tube) to a liter or more (e.g., several thousand liters). In various embodiments, no vapor diffusion process is involved. In various embodiments, no evaporation is involved. In various embodiments, batch crystallization involves slowly adding a precipitating reagent (e.g., with stirring, if necessary, depending on the batch size).

[0106] Typically, seeds of crystallization material can be added early in the batch process to improve reproducibility and product quality. Once a desired amount of solids has been formed, the slurry is typically transferred to a solid-liquid separation unit.

[0107] Dialysis is another method commonly used for crystallizing polypeptides. This technique uses the diffusion and equilibration of precipitating molecules through a semipermeable membrane as a means of gradually approaching the concentration at which the macromolecule will crystallize. When large amounts of polypeptide are available, dialysis tubing can be used.

[0108] Microdialysis buttons, also known as Cambridge buttons, provide a convenient way to produce crystals from small sample volumes. A polypeptide sample is placed in a small chamber on top of the button, and the sample is covered with a dialysis membrane of appropriate molecular weight cutoff. The device is then immersed in a reservoir containing a precipitant solution. Equilibration of the precipitant molecules is allowed to occur across the membrane.

[0109] Free interface diffusion can also be used to crystallize polypeptides. This technique involves carefully layering a precipitant solution on top of a concentrated polypeptide solution in a capillary, which is sealed at both ends with wax. The small diameter of the capillary minimizes mixing from natural convection in the system. Thus, the precipitant and polypeptide slowly interdiffuse and the system reaches equilibrium by a phenomenon called counterdiffusion. When the solutions first come into contact and diffusive mixing occurs, the region of the polypeptide solution near the interface becomes supersaturated, creating ideal conditions for nucleation. As time progresses, the two solutions interdiffuse along the axis of the capillary, diluting each other, thus promoting the dissolution of smaller nuclei and the growth of larger nuclei. Free liquid diffusion achieves transient nucleation conditions, which in most cases allows high quality crystals to be obtained. Thus, free interface diffusion can be considered as a rational crystallization approach to minimize supersaturation and impurity levels at the crystal growth front and ensure stability of both values. One variation of the free interface diffusion method is called the liquid bridge method, in which a drop of a polypeptide sample and a drop of a precipitant solution are placed in close proximity on a cover slip and connected by a thin liquid bridge. The liquid diffusion between the two drops is sealed from the air and can induce crystal growth.

[0110] In some cases, crystallization nucleation can be induced by using materials such as nucleating agents, nucleants, or seeds. Nucleation can occur at the surface of the nucleating agents, nucleants, or seeds, thereby inducing an increase in the local concentration of macromolecules, lowering the energy barrier of nucleation, and avoiding the kinetic barrier of spontaneous nucleation. Supersaturation may be required under these circumstances.

[0111] Pharmaceutical cocrystals are crystalline materials composed of a pharma- ceutical active ingredient and one or more co-crystal formers ("coformers"), such that the active ingredient and the co-formers exist together in the same crystal lattice. Cocrystals are distinct from salts because, unlike salts, the components that coexist in the co-crystal lattice with a defined stoichiometry interact non-ionically. Cocrystals are also distinct from polymorphs, which are defined as including multi-component phases, such as 1) single-component crystalline forms with different molecular arrangements or conformations in the crystal lattice, 2) amorphous, and 3) solvated and hydrated forms. Cocrystals are similar to solvates, at least in that both contain multiple components in the lattice. In some embodiments, the amorphous or crystallized polypeptide compositions of the present disclosure do not include a co-crystallized polypeptide. In some embodiments, the amorphous or crystallized polypeptide compositions of the present disclosure include a co-crystallized polypeptide.

[0112] Those skilled in the art will understand that many crystallization conditions can be adjusted to increase or decrease the efficiency and / or purity of crystallization. For example, crystallization conditions that can be adjusted include solubilization system (aqueous and / or organic solvent system), pH, counterions, salts, temperature, excipients, coformers, and polypeptide concentration. It is simple for those skilled in the art to adjust these factors and test their ranges. In the art of crystallization, it is considered typical to sample a wide variety of conditions. Furthermore, as those skilled in the art will understand, the present disclosure does not necessarily require identifying the most efficient crystallization form, only that crystals can be formed.

[0113] The crystallized polypeptide composition of the present disclosure may comprise a plurality of crystallized polypeptides. In various embodiments, the crystallized polypeptide may be characterized by high concentration, high purity, and / or high stability. Any suitable method known in the art may be used to characterize the provided crystallized polypeptide composition, including, but not limited to, X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), high performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LCMS), laser diffraction, hot stage microscopy, polarized light microscopy, and the like.

[0114] In various embodiments, the amorphous or crystallized polypeptide compositions of the present disclosure are free or substantially free of one or more or any precipitation reagents, optionally the precipitation reagent comprises one or more of a salt, an organic solvent, or a polymer, optionally the salt can comprise a salt selected from ammonium sulfate, citrate salt, and cetyltrimethylammonium salt, the organic solvent can comprise 2-methyl-2,4-pentanediol or an organic solvent selected from 2-methyl-2,4-pentanediol, and / or the polymer can comprise a polymer that is polyethylene glycol.

[0115] In various embodiments, the amorphous or crystallized polypeptide compositions of the present disclosure comprise polypeptide crystals that are free or substantially free of one or more or any precipitation reagents, where optionally the precipitation reagent comprises one or more of a salt, an organic solvent, or a polymer, where optionally the salt can comprise a salt selected from ammonium sulfate, citrate, and cetyltrimethylammonium salt, the organic solvent can comprise 2-methyl-2,4-pentanediol or an organic solvent selected from 2-methyl-2,4-pentanediol, and / or the polymer can comprise a polymer that is polyethylene glycol.

[0116] Unless otherwise specified, those of skill in the art will understand that references to crystallization in the art refer to crystallization of molecules that are not polypeptides, e.g., crystallization of molecules that are small molecules (e.g., small molecule therapeutic agents).

[0117] In various applications, peptide crystallization can obtain ordered crystals with generally uniform content.Although the present disclosure includes polypeptide compositions that include ordered crystals, it also specifically recognizes that the formulations of the present disclosure do not require crystals with specific or consistent size or characteristics.In various embodiments, crystallized polypeptide compositions include one or more crystalline forms of polypeptide (e.g., one or more polymorphs or hydrates of polypeptide).

[0118] In some embodiments, the polypeptide / peptide can be in an aqueous form in the form of droplets coated with vitamin E. In other embodiments, derivatives of vitamin E that can be dissolved in aqueous solutions can be used as carriers.

[0119] Those of skill in the art will appreciate from the present disclosure that the compositions and methods provided herein are useful for the delivery of a wide variety of polypeptides. For example, those of skill in the art will appreciate from the present disclosure that the compositions and methods provided herein are advantageous, at least in part, because they deliver polypeptides, e.g., via the lymphatic system, to the bloodstream and / or plasma following oral administration, e.g., due to advantageous pharmacokinetic properties.

[0120] In some embodiments, the polypeptide has a molecular weight that can be, for example, at least about 25 kDa, 50 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 400 kDa, 500 kDa, 1,000 kDa, or more. The polypeptide of the present disclosure can have a molecular weight in the range of, for example, about 1 to about 1,000 kDa, for example, about 25 kDa to about 1,000 kDa, about 25 kDa to about 500 kDa, about 100 kDa to about 500 kDa, about 125 kDa to about 250 kDa, about 125 kDa to about 175 kDa, or about 150 kDa to about 300 kDa. In various embodiments, the polypeptides of the disclosure can have a molecular weight with a lower limit, e.g., of about 1, 10, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 kDa, and an upper limit, e.g., of about 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, or 1,000 kDa.

[0121] In some embodiments, the polypeptide has a molecular weight that can be, for example, about 50 Da to about 25 kDa, and optionally the molecular weight is between about 50 Da to about 1 kDa, between about 50 Da to about 2 kDa, between about 50 Da to about 3 kDa, between about 50 Da to about 4 kDa, between about 50 Da to about 5 kDa, between about 50 Da to about 10 kDa, between about 50 Da to about 15 kDa, or between about 50 Da to about 20 kDa. In various embodiments, the polypeptides of the disclosure may have a molecular weight with a lower limit of, e.g., about 50 Da, about 100 Da, about 150 Da, about 200 Da, about 250 Da, about 300 Da, about 400 Da, about 500 Da, about 600 Da, about 700 Da, about 800 Da, about 900 Da, about 1 kDa, about 2 kDa, about 3 kDa, about 4 kDa, or about 5 kDa, and an upper limit of, e.g., about 250 Da, 300 Da, 400 Da, 500 Da, 600 Da, 700 Da, 800 Da, 900 Da, 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa, or 25 kDa.

[0122] A polypeptide of the present disclosure may contain, for example, about 1 to about 1,000 amino acids, such as about 10 to about 500, about 10 to about 250, about 10 to about 100, about 100 to about 1,000, about 100 to about 750, about 100 to about 500, about 100 to about 250, about 250 to about 1,000, about 250 to about 750, or about 250 to about 500 amino acids. In various embodiments, polypeptides of the disclosure may include a number of amino acids having a lower limit of, e.g., about 1, 10, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids and an upper limit of, e.g., about 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, or 1,000 amino acids.

[0123] In various embodiments, the polypeptides of the present disclosure can be therapeutic polypeptides, e.g., enzymes or antibodies, for administration to a subject in need thereof. One of skill in the art would be familiar with the identity and properties of a wide variety of therapeutic polypeptides.

[0124] In various embodiments, the polypeptides of the disclosure can be or can include recombinant polypeptides, isolated or synthetic polypeptides, cytoskeletal proteins, extracellular matrix proteins, plasma proteins, clotting factors, acute phase proteins, heme proteins, cell adhesion proteins, transmembrane transport proteins, symport / antiport proteins, hormones, growth factors, receptors, transmembrane receptors, intracellular receptors, DNA binding proteins, transcriptional regulatory proteins, RNA binding proteins, immune system proteins, nutrient storage and transport proteins, chaperone proteins, enzymes, glycoproteins, phosphoproteins, membrane proteins, transport proteins, or lipoproteins, antibodies, recombinant antibodies, antibody fragments, monoclonal antibodies, modified enzymes, pegylated polypeptides, therapeutic polypeptides, storage polypeptides, enzymes, growth factors or hormones, immunomodulatory agents, anti-infective agents, anti-proliferative agents, vaccines, or other therapeutic, prophylactic, diagnostic polypeptides, and combinations thereof.

[0125] In various embodiments, the polypeptides of the present disclosure may be or may include an antibody or an antibody fragment. In some embodiments, the antibody is a monoclonal antibody or a fragment thereof. In some embodiments, the antibody is a polyclonal antibody or a fragment thereof. In some embodiments, the antibody is natural, synthetic, or engineered.

[0126] Antibody fragments include Fab fragments (a single Fab isolated from an intact antibody by digestion with the enzyme papain) and F(ab')2 fragments (two Fabs covalently linked together, produced by digesting an antibody with the enzyme pepsin). Fab fragments are monospecific, while F(ab')2 fragments are bispecific. Antibody fragments include double-chain Fv (dsFv) fragments and single-chain Fv (scFv) fragments (the "v" stands for "variable" in both cases). dsFv fragments consist of a Fab fragment minus the constant regions, i.e., they consist only of the variable regions of the heavy and light chains covalently linked together. scFv fragments are a single polypeptide chain, consisting of the variable region of the heavy chain linked to the variable region of the light chain via a peptide linker. Classically, both dsFv and scFv fragments are monovalent (and therefore monospecific). However, two dsFv or two scFv fragments can be joined to form a bispecific fragment (which would be similar to a F(ab')2 fragment without the constant region). In addition, two dsFv or scFv fragments with different antigen binding sites (i.e., different specificities) can be joined to form a bispecific fragment. Such fragments can be used as research tools or as therapeutic or diagnostic reagents.

[0127] In some embodiments, the antibody is an immunoglobulin. In naturally occurring immunoglobulins, each tetramer comprises two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The amino-terminal portion of each chain contains a variable ("V") region of about 100-110 or more amino acids that are primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region that is primarily responsible for effector function. The four chains are arranged in a classical "Y" model. The bottom "leg" of the "Y" is called the Fc region ("c" stands for "crystallizable" or "complement-binding") and is used to anchor the antibody in the cell membrane and also to bind and activate complementation with macrophage cells. The two "arms" at the top of the "Y" are called the Fab region ("ab" stands for "antigen-binding"). Each Fab region contains a constant region (the junction of the Fab and Fc regions) and a variable region (extending to the end of the "Y" or Fc region). Each variable region contains identical antigen binding sites at each end of the "Y" (regions within the variable region termed "hypervariable" regions). The term "hypervariable" region refers to the complementarity determining regions or CDRs (i.e., amino acid residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). "Framework" or FR residues are the remaining variable region residues other than the hypervariable region residues. Each Fab region has one antigen-binding site, such that the intact antibody molecule has two antigen-binding sites (i.e., is "bivalent"). Because the two antigen-binding sites on a naturally occurring antibody are identical to each other, the antibody is specific for one antigen (i.e., is "monospecific").

[0128] Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the constant domain of the heavy chain. Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε), which define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Typically, IgG, IgE, and IgD occur as monomers, while IgA can occur not only as monomers but also as dimers or trimers, and IgM can occur as a pentamer. Some of the above can be further classified into subclasses or isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Different isotypes have different effector functions, for example, IgG1 and IgG3 isotypes have antibody-dependent cellular cytotoxicity (ADCC) activity. Human light chains are classified as kappa (κ) light chains and lambda (λ) light chains. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain further including a "D" region of about 10 more amino acids (see generally, Fundamental Immunology, Ch. 7 (Paul, W. ed., 2nd ed. Raven Press, NY (1989)).

[0129] In some embodiments, the polypeptide may be a chimeric antibody. While naturally occurring antibodies are derived from a single species, engineered antibodies and antibody fragments may be derived from multiple animal species, i.e., chimeric. Mouse (murine) / human chimeric antibodies have been created, but other combinations are possible. Chimeric antibodies are further classified into two subtypes: chimeric antibodies and humanized antibodies. Typically, mouse / human chimeric antibodies contain approximately 75% human amino acid sequences and approximately 25% mouse amino acid sequences. The human sequences correspond to the constant regions of the antibody, and the mouse sequences correspond to the variable regions of the antibody (and thus contain the antigen binding site). The rationale for using such chimeras is to retain the antigen specificity of the mouse antibody, but reduce the immunogenicity of the mouse antibody (mouse antibodies provoke an immune response against them in species other than mice), allowing the chimeras to be used in human therapy. Chimeric antibodies also include antibodies that contain CDR regions of different human antibodies. CDR regions, also called hypervariable regions, are sequences within the variable regions of an antibody molecule that form the antigen binding site. The CDR regions are so named because the binding site is complementary in shape and charge distribution to the epitope recognized on the antigen. Alternatively, chimeric antibodies contain framework regions from one antibody and CDR regions from another antibody. Chimeric antibodies also include antibodies that contain the CDR regions of at least two different human antibodies. Typically, humanized antibodies contain approximately 90% (or more) human amino acid sequences. In this scenario, the only mouse sequences present are those of the hypervariable regions (which are the actual antigen binding sites contained within the variable regions). Humanized antibodies have minimal mouse immunogenicity compared to chimeric antibodies.

[0130] Examples of antibodies and antibody fragments include, but are not limited to, idarucizumab (Praxbind®), raxibacumab (ABSTRAX®), atezolizumab (TECENTRIQ®, RG7446 (Roche)), ofatumumab (Arzerra®), obinutuzumab (GAZYVA®, GA101 (Roche)), bezlotoxumab (ZINPLAVA™), necitumumab (Portrazza™), obiltoxaximab (ANTHIM®), olaratumab (Lartruvo™), rituximab (RITUXAN®, ABP 798 (Amgen), MabThera®, GP2013 (Novartis)), tositumomab (Bexxar®), trastuzumab (HERCEPTIN®, ABP 980 (Amgen), HERTRAZ™, CANMAB™), pertuzumab (PERJETA®, RG1273 (Roche)), tocilizumab (ACTEMRA®), bevacizumab (AVASTIN®, ABP 215 (Amgen), daratumumab (Darzalex®), elotuzumab (EMPLICITI™), siltuximab (SYLVANT™), panitumumab (Vectibix®), vedolizumab (Entyvio®), eculizumab (Soliris®), natalizumab (TYSABRI®), cetuximab (ERBITUX®), ipilimumab (YERVOY®), reslizumab (CINQAIR®), pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), infliximab (REMICADE®, ABP710 (Amgen), FLIXABI®), abciximab (ReoPro®), evolocumab (Repatha®), secukinumab (Cosentyx®), certolizumab pegol (Cimzia®), ixekizumab (TALTZ™), omalizumab (Xolair®), canakinumab (Ilaris®), alirocumab (Praluent®), daclizumab ramucirumab (CYRAMZA®), ramucirumab (ZINBRYTA™, ZENAPAX®), denosumab (XGEVA®), denosumab (Prolia®), mepolizumab (Nucala), ustekinumab (Stelara®), golimumab (Simponi®), adalimumab (HUMIRA®, ABP501 (Amgen), GP2017 (Novartis)), ramucirumab (CYRAMZA®), ranibizumab ( (LUCENTIS®, RG3645 (Roche & Novartis)), efalizumab (Raptiva®), palivizumab (Synagis®), Ado-trastuzumab emtansine (KADCYLA™), alemtuzumab (Campath®), alemtuzumab (LEMTRADA™), basiliximab (Simulect®), belimumab (Benlysta®) , blinatumomab (BLINCYTO®), brentuximab vedotin (Adcetris), capromab pendetide (ProstaScint®), dinutuximab (Unituxin), elotuzumab (EMPLICITI™), gemtuzumab ozogamicin (Mylotarg), ibritumomab tiuxetan (Zevalin®), itolizumab (Alzumab™), muromonab (OrthocloneOKT3®), nimotuzumab (Theracim®), nofetumomab (Verluma®), and biosimilars and combinations thereof. In various embodiments, examples of antibodies and antibody fragments include, but are not limited to, abciximab, palivizumab, murumonab-CD3, gemtuzumab, trastuzumab, basiliximab, daclizumab, etanercept, ibritumomab, and / or tiuxetan.

[0131] Examples of antibodies and antibody fragments include, but are not limited to, anti-cytokine antibodies, anti-CD antigen antibodies (e.g., anti-CD3, -CD20 (rituximab), anti-CD25, anti-CD52, anti-CD33, and anti-CD11a), anti-TNF-α (e.g., infliximab), anti-rattlesnake venom, anti-ICAM (e.g., anti-ICAM-1 and anti-ICAM-3), anti-growth factor antibodies (e.g., anti-VEGF), anti-growth factor receptor antibodies (e.g., anti-HER2 / neu (trastuzumab) and anti-EGFR), anti-immunoglobulin antibodies (e.g., anti-IgE), anti-polyclonal Ab antibodies. , antiviral antibodies (e.g., anti-CMV, anti-HIV (anti-gp120), anti-HBV, anti-RSV (anti-F glycoprotein)), anti-complement antibodies (e.g., anti-C5), anticoagulant antibodies (e.g., anti-gpIIb / IIIa and anti-factor VII), anti-interleukin antibodies (e.g., anti-IL-5, anti-IL-4, and anti-IL-8), antibodies targeting major histocompatibility complexes (e.g., anti-HLA), anti-idiotypic antibodies, anti-integrin antibodies (e.g., anti-β-2-integrin), anti-17-IA cell surface antigen, anti-α4β7, anti-VLA-4, anti-CBL, and combinations thereof.

[0132] In some embodiments, the polypeptide is a biosimilar antibody. Generally, biosimilars are physiochemically or biologically similar to the reference, both in terms of safety and efficacy. Biosimilars can be evaluated against the reference using one or more in vitro tests. In vitro comparisons can be combined with in vivo data showing similarity in pharmacokinetics, pharmacodynamics, and / or safety. Clinical evaluations can include comparison of pharmacokinetic properties (e.g., AUC0-inf, AUC0-t, Cmax, tmax, Ctrof), pharmacodynamic endpoints, or similarity in clinical efficacy (e.g., using randomized parallel-group controlled clinical trials). Differences between the biosimilar and the reference can include post-translational modifications, such as conjugation of one or more biochemical groups (e.g., phosphates, various lipids, and carbohydrates), post-translational proteolytic cleavage, changes in the chemical nature of amino acids (e.g., formylation), or many other mechanisms. Other post-translational modifications can be the result of manufacturing process operations. For example, glycation can occur when the product is exposed to reducing sugars. In other cases, storage conditions may permit certain degradation pathways, such as oxidation, deamidation, aggregation, etc.

[0133] Generally, antibodies can be used to treat, for example, cancer, inflammation, cardiovascular disease, and transplant rejection due to their specific target binding properties and / or target neutralization, e.g., binding and / or neutralization of targets associated with disease states. For example, the monoclonal antibody infliximab binds to tumor necrosis factor and neutralizes its role in inflammation by blocking its interaction with cell surface receptors. Rituximab targets malignant B lymphocytes by binding to their cell surface CD20 antigen. Clinically relevant antibodies may also be classified according to the therapeutic area in which they are used. In some embodiments, clinical antibodies used for therapeutic use may include antibodies for treating cancer (e.g., pancreatic cancer), inflammatory diseases (e.g., autoimmune diseases, arthritis), cardiovascular diseases (e.g., stroke), infectious diseases (e.g., HIV / AIDS), respiratory diseases (e.g., asthma), tissue transplant rejection, and organ transplant rejection. In some embodiments, clinical antibodies are used in radioimmunotherapy.

[0134] In various embodiments, the polypeptides of the disclosure can be or include polypeptide replacement therapies, enzymes (e.g., therapeutic replacement enzymes), and / or fusion polypeptides. Examples of polypeptides and / or enzymes include, for example, antibodies (e.g., anti-HER2 antibodies, e.g., trastuzumab), GLP-1 receptor agonists, human glucagon-like peptide-1 (GLP-1) or analogs thereof (e.g., synthetic analogs, e.g., liraglutide), parathyroid hormone or analogs thereof (e.g., recombinant human parathyroid hormone analogs, e.g., teriparatide), insulin or analogs thereof (e.g., Humulin®), rituximab, bevacizumab, cetuximab, etanercept, infliximab, or analogs or derivatives thereof. Examples of enzymes, e.g., enzymes for replacement enzyme therapy, include agalsidase beta, agalsidase alpha, imiglucerase, aliglucerase alpha, velaglucerase alpha, alglucerase, sebelipase alpha, laronidase, idursulfase, elosulfase alpha, galsulfase, pancrelipase, sapropterin, eliglustat, galsulfase, asfotase alpha, pegvalase, elapegademase, and / or sacrosidase. Examples of replacement polypeptides include factor I, factor II, factor III, factor IV, factor V, factor VI, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, and / or von Willebrand factor. Examples of fusion polypeptides can include, for example, etanercept, aflibercept, rilonacept, alefecept, romiplostim, abatacept / belatacept, and / or denileukin-diftitox. The disclosure includes analogs and modified forms of the polypeptides disclosed herein, including, for example, one or more pendant groups or modifications, such as pegylation, acetylation, amidation, lipidation, methylation, phosphorylation, glycosylation, glycosylation, sulfation, mannosylation, nitrosylation, acylation, palmitoylation, prenylation, or combinations thereof.In various specific embodiments, the polypeptides of the present disclosure comprise one or more pendant groups or modifications selected from pegylation and / or fatty acids.

[0135] The present disclosure further includes engineered and / or biosimilar forms of the polypeptides disclosed herein, e.g., having at least 80% sequence identity to the polypeptides disclosed herein, e.g., having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the polypeptides disclosed herein.

[0136] The present disclosure further includes the recognition that polypeptides (including, e.g., antibodies and / or antibody fragments) are not generally referred to in the art as "small molecules" or "compounds." Thus, because small molecules and compounds may have very different physical and pharmacokinetic properties than the polypeptides (e.g., antibodies and / or antibody fragments) disclosed herein, one of skill in the art would not understand that prior art disclosures relating to small molecules and compounds are applicable to formulations and methods relating to the polypeptides disclosed herein.

[0137] Pharmaceutically acceptable capsules The core comprising the amorphous or crystallized polypeptide composition of the present disclosure can be encapsulated in a polymer capsule. The capsule of the present disclosure includes a pharma- ceutically acceptable capsule suitable for oral or rectal administration. In various embodiments, the capsule of the present disclosure is a capsule suitable for delivery (e.g., specific delivery) of a polypeptide to the intestine. In various embodiments, the capsule of the present disclosure is a capsule suitable for delivery (e.g., specific delivery) of a polypeptide to one or both of the small intestine and / or the large intestine.

[0138] The capsules of the present disclosure include hard-shell capsules and soft-shell capsules. In various embodiments, the capsule is a gelatin capsule or a hydroxypropylmethylcellulose (HPMC) capsule. Exemplary capsule materials can include polymers such as polyglycolic acid (PGA), polylactic acid (PLA) and its copolymers, poly(lactide-co-glycolide) (PLGA), and non-ionic cellulose ethers such as hydroxypropylcellulose (HPC) and hydroxypropylmethylcellulose (HPMC). In some embodiments, the capsule is a vegetable capsule.

[0139] Soft gelatin capsules, also called softgels, can be made from a single piece of gelatin. They can be used to encapsulate non-water-based solutions (because water dissolves gelatin). When ingested, the capsule dissolves, exposing the core.

[0140] Hard gelatin capsules are made of two parts: a body and a cap. They can be filled with a core that contains dry ingredients in powder form. The body is first filled with the core composition, and then the capsule is closed with a cap. When ingested, the hard capsule dissolves, exposing the core.

[0141] In various embodiments, the capsule may include an enteric coating. Typically, an enteric coating is a barrier that controls the location in the recipient body (e.g., the digestive system, e.g., the intestine, e.g., the small intestine and / or the large intestine) to which the oral formulation core is exposed and / or delivered. Many enteric coatings are insoluble at low pH, but dissolve, swell, or solubilize at the high pH in the intestinal tract. Typical materials used for enteric coatings can include CAP (cellulose acetate phthalate), CAT (cellulose acetate trimellitate), PVAP (poly(vinyl acetate phthalate)), and HPMCP (hydroxypropyl methylcellulose phthalate), poly(methacrylic acid-co-methyl methacrylate), fatty acids, waxes, shellac (e.g., esters of aryl acids), plastics, and vegetable fibers. In various embodiments, the dissolution pH of the enteric coating can be, for example, between about 4.5 and about 7, e.g., about 4.5 to about 5.5, about 4.5 to about 6.0, 5.5 to about 7.0, about 5.0, about 6.2, or about 7.0. In various embodiments, the enteric coating has a pH release within a range having a lower limit of about 4.5, 5.0, 5.5, or 6.0 and an upper limit of about 5.0, 5.5, 6.0, 6.5, 7.0, or 7.5.

[0142] The present disclosure expressly includes the recognition that enteric coating is not required for delivery of the polypeptides disclosed herein in accordance with the methods and compositions of the present disclosure to reach the blood stream, plasma, lymphatic system, and / or thoracic duct, e.g., in therapeutically effective amounts.

[0143] In various embodiments, the formulations of the present disclosure that include amorphous or crystallized polypeptide compositions are bioadhesive formulations or include them, for example, in or on capsules or tablets. In various embodiments, the bioadhesive formulations adhere to specific biological locations, such as mucosal linings (mucoadhesion). Bioadhesive dosage forms can improve oral absorption of polypeptide drugs by delivering small doses over long periods of time and / or localizing them in the intestine through bioadhesion. Various bioadhesive polymers can be broadly specific or nonspecific. Specific bioadhesive polymers (e.g., lectins and fimbrins) have the ability to adhere to specific chemical structures within biomolecules, whereas nonspecific bioadhesive polymers (polyacrylic acid [PAA] and cyanoacrylates) have the ability to bind to both cell surfaces and mucosal layers. Further examples of bioadhesive polymers include sodium CMC, carbopol, polycarbophil, tragacanth, sodium alginate, HPMC, karaya gum, gelatin, guar gum, pectin, gum arabic, chitosan, and hydroxypropyl cellulose. Examples of bioadhesive polymers include hydrophilic polymers (e.g., methylcellulose, hydroxyethylcellulose, HPMC, Na CMC, and carbomer), thiolated polymers (e.g., chitosan-iminothiolane, PAA-cysteine, PAA-homocysteine, chitosan-thioglycolic acid, chitosan-thioethylamidine, alginic acid-cysteine, poly(meth)acrylic acid-cysteine, sodium carboxymethylcellulose-cysteine), lectin-based polymers (e.g., lentil lectin, peanut agglutinin, and ulex europaeus agglutinin), Polyox WSR (e.g., WSR N-10, WSR N-80, WSR N-205, and WSR N-750), and other polymers (e.g., tomato lectin, PAA-co-PEG, and PSA). The present disclosure further includes suppositories suitable for rectal delivery. In various embodiments, the suppository melts or dissolves upon administration.In various embodiments, the suppository includes a capsule (e.g., a capsule disclosed herein, e.g., a hard gelatin capsule or a soft gelatin capsule) that encloses the core. In various embodiments, the suppository does not include a capsule (i.e., does not include a capsule that encloses the core). The suppository can be formed from a waxy substance, structured glycerin, hydrogenated vegetable oil, polyethylene glycol wax derivative, or a poloxamer-based mixture. In various embodiments, the suppository is solid at ambient temperature but melts rapidly at body temperature. An emulsifier can be used to increase the solubility of the crystallized or amorphous polypeptide in the suppository mass and / or to facilitate the dispersion of the crystallized or amorphous polypeptide after the suppository melts.

[0144] In various embodiments, the present disclosure further includes tablets, fast dissolving tablets, liquid gel capsules, syrups, lozenges, aerosol sprays, chewing gums, and the like.

[0145] formulation The disclosure includes methods and compositions comprising a polypeptide formulation for oral or rectal delivery comprising a core within a pharma- ceutically acceptable capsule or suspension, the core or suspension comprising a polypeptide composition (e.g., an amorphous polypeptide composition or a crystallized polypeptide composition) and a pharma- ceutically acceptable carrier comprising a vitamin E agent.

[0146] In various embodiments, the suspensions of the present disclosure comprise a suspension of an amorphous or crystallized polypeptide composition in a vitamin E agent and / or a pharma- ceutically acceptable carrier comprising a vitamin E agent. The suspensions of the present disclosure comprise a liquid formulation in which an amorphous or crystallized polypeptide drug is distributed in a suspension medium (e.g., a vitamin E agent, a pharma- ceutically acceptable carrier, or a combination thereof). In various embodiments, the suspensions of the present disclosure can be dilute suspensions (e.g., a suspension comprising 10% or less polypeptide drug by weight or volume) or concentrated suspensions (e.g., a suspension comprising 10-50% or more polypeptide drug by weight or volume). In various embodiments, the suspensions of the present disclosure can be aggregated or disaggregated suspensions. In various embodiments, the suspensions can be coarse suspensions (e.g., a suspension of particles greater than 1 μm in diameter), colloidal suspensions (e.g., a suspension of particles less than 1 μm in diameter), or nanosuspensions (e.g., a suspension of particles 1-100 nm in diameter). The suspensions of the present disclosure can be administered, for example, orally or rectally.

[0147] In various embodiments, the core or suspension comprises about 1 mg to 2,000 mg of polypeptide (e.g., crystallized or amorphous polypeptide). In various embodiments, the core or suspension comprises about 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 400 mg, 1 mg to 300 mg, 1 mg to 200 mg, or 1 mg to 100 mg of crystallized or amorphous polypeptide. In some embodiments, the formulation comprises an amount of crystallized or amorphous polypeptide having a lower limit of, e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg of crystallized or amorphous polypeptide and an upper limit of 100, 150, 200, 250, 300, 350, 400, 450, 500, 1,000, or 2,000 mg. In various embodiments, the core or suspension comprises between about 50 μg and 2,000 mg of polypeptide, and optionally the polypeptide formulation comprises between about 50 μg and 1,000 mg, 50 μg and 500 mg, 50 μg and 400 mg, 50 μg and 300 mg, 50 μg and 200 mg, 50 μg and 100 mg, 50 μg and 50 mg, 50 μg and 25 mg, 50 μg and 20 mg, 50 μg and 15 mg, 50 μg and 10 mg, 50 μg and 5 mg, 50 μg and 1 mg, 50 μg and 500 μg, 1 mg and 1,000 mg, 1 mg and 500 mg, 1 mg and 400 mg, 1 mg and 300 mg, 1 mg and 200 mg, 1 mg and 100 mg, 1 mg and 50 mg, 1 mg and 25 mg of crystallized or amorphous polypeptide. In various embodiments, the core or suspension comprises between about 1 μg and 2,000 mg of polypeptide, and optionally the polypeptide formulation comprises between about 1 μg and 1,000 mg, 1 μg and 500 mg, 1 μg and 400 mg, 1 μg and 300 mg, 1 μg and 200 mg, 1 μg and 100 mg, 1 μg and 50 mg, 1 μg and 25 mg, 1 μg and 20 mg, 1 μg and 15 mg, 1 μg and 10 mg, 1 μg and 5 mg, 1 μg and 1 mg, 1 μg and 500 μg, 1 μg and 250 μg, 1 μg and 200 μg, 1 μg and 150 μg, 1 μg and 100 μg, 1 μg and 50 μg of crystallized or amorphous polypeptide.In some embodiments, the formulation comprises an amount of crystallized or amorphous polypeptide having a lower limit of, e.g., 1 μg, 5 μg, 10 μg, 15 μg, 20 μg, 25 μg 50 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, or 25 mg of crystallized or amorphous polypeptide and an upper limit of, e.g., 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 25 mg, 50 mg, 100 mg, 500 mg, 1,000 mg, or 2,000 mg of crystallized or amorphous polypeptide.

[0148] In various embodiments, the formulations (e.g., encapsulated or suspension formulations) of the present disclosure and / or their cores comprise a molar or weight excess of the vitamin E agent relative to the amount of crystallized or amorphous polypeptide in the formulation, optionally where the excess is a fold excess of at least 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, or 1,000 times, e.g., a fold increase in the range of 1-10, 1-20, 1-30, 1-40, 1-50, 1-100, or 1-1,000 times. In various embodiments, the molar or weight excess of the vitamin E agent relative to the amount of crystallized or amorphous polypeptide in the formulation is a fold increase within a range having a lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 75, 100, 150, 200, or 250 and an upper limit of 30, 40, 50, 75, 100, 150, 200, 250, 500, or 1,000.

[0149] In various embodiments, one or more pharma- ceutically acceptable carriers are present in the formulations (e.g., encapsulated or suspended formulations) or their cores of the present disclosure at a concentration of between 0.1 mM and about 1,000 mM, between about 0.1 mM and about 500 mM, between about 0.1 mM and about 200 mM, or between about 1 mM and about 100 mM. In various embodiments, one or more pharma- ceutically acceptable carriers are present in the formulations (e.g., encapsulated or suspended formulations) or their cores of the present disclosure at a concentration having a lower limit of 0.1, 1, 5, 10, 20, 30, 40, 50, 75, or 100 mM and an upper limit of 10, 20, 30, 40, 50, 75, 100, 150, 200, 250, or 500 mM.

[0150] Certain suppository formulations of the present disclosure may include an amorphous or crystallized polypeptide composition and a suppository excipient, such as a lipophilic base (e.g., cocoa butter, coconut oil, virgin coconut oil, almond oil, wheat germ oil, any edible oil, hydrogenated vegetable oil, and hard fat) or a hydrophilic base (e.g., glycerinated gelatin and polyethylene glycol). Lipophilic bases are immiscible with body fluids and melt easily at body temperature to release the drug on the mucosal surface, whereas hydrophilic bases need to dissolve in physiological fluids to release the drug. Suppository formulations may include solid, semi-solid, and liquid forms.

[0151] In various embodiments, the suppository formulation of the present disclosure comprises a semi-solid dosage form such as a gel or foam. Rectal gels are semi-solid formulations that entrap solvents within a polymer network to create a viscous consistency. The viscosity of the gel can be modified by adding co-solvents (e.g., glycerin and propylene glycol) and electrolytes.

[0152] In various embodiments, the suppository formulations of the present disclosure include liquid suppositories, such as those comprising a thermosensitive polymer (e.g., poloxamer), a mucoadhesive polymer (e.g., carbopol, sodium alginate, polycarbophil, hydroxypropylmethylcellulose, hydroxyethylcellulose, and methylcellulose), or a combination of a thermosensitive polymer and a mucoadhesive polymer. The suppository may further comprise, for example, a cellulose ether polymer (e.g., hydroxypropylmethylcellulose, hydroxyethylcellulose, and methylcellulose).

[0153] In various embodiments, the suppository formulations of the present disclosure include foams, e.g., colloidal dosage forms having a hydrophilic liquid continuous phase containing a foaming agent and a gaseous dispersed phase dispersed throughout. After rectal administration, certain such formulations transition from a foam state to a liquid or semi-solid state on the mucosal surface. The foaming agent includes an amphiphilic substance that is important for foam generation and stabilization.

[0154] In addition to the above, the pharmaceutical composition of the present disclosure may take any form known in the art. Such forms include, for example, liquid, semi-solid, and solid dosage forms, such as solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The pharmaceutical composition of the present disclosure may be administered enterally or topically, for example bucally. The pharmaceutical composition of the present disclosure may take the form of an injectable or infusible solution. The pharmaceutical composition of the present disclosure may be formulated for administration by parenteral modes (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection). As used herein, parenteral administration refers to modes of administration other than enteral and topical administration, such as by injection, including, but not limited to, intravenous, intranasal, intranasal, intraocular, pulmonary, intrapulmonary, extrapulmonary, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intraperitoneal, transtracheal, subcutaneous, transdermal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid, and intrasternal injection and infusion. The pharmaceutical composition can be administered parenterally in the form of an injectable formulation, including a sterile solution or suspension. For example, the pharmaceutical composition can be formulated by suitably combining the polypeptide composition with one or more pharma- ceutical acceptable carriers. Administration can be systemic or local.

[0155] In some embodiments, the composition can be formulated for pulmonary administration (e.g., administration via an inhaler or nebulizer). Methods for formulating such compositions are well known in the art. Pulmonary administration can be oral and / or nasal. Examples of pharmaceutical devices for pulmonary delivery include metered dose inhalers, dry powder inhalers (DPIs), and nebulizers.

[0156] In some embodiments, the composition can be formulated for delivery to the eye, for example, in the form of a pharma- ceutically acceptable solution, suspension, or ointment (e.g., eye drops). The formulations used for treating the eye can be in the form of a sterile aqueous solution, including, for example, but not limited to, additional components such as preservatives, buffers, tonicity adjusting agents, antioxidants and stabilizers, non-ionic wetting or clarifying agents, and viscosity increasing agents. The formulations described herein can be administered topically to the eye of a subject in need of treatment in a conventional manner, for example, in the form of drops or by immersing the eye in the treatment solution.

[0157] Suitable administration means can be selected based on the age and condition of the subject. The amount of active ingredient contained in the pharmaceutical preparation is such that a suitable dose within the specified range is provided. The dose and administration method vary according to the patient's weight, age, condition and other characteristics, and can be suitably selected by those skilled in the art according to the need.

[0158] Polypeptide delivery and applications The compositions and methods of the present disclosure include formulations for oral administration to a subject, which results in delivery of a polypeptide to the subject's thoracic duct, lymphatic system, and / or bloodstream. Without wishing to be bound by any particular scientific theory, knowledge of the inventors, the present disclosure is the first to utilize oral formulations and / or oral formulations containing vitamin E agents to deliver a polypeptide to the bloodstream via the thoracic duct and / or lymphatic system.

[0159] For a long time, it was assumed that vitamin E was absorbed by passive diffusion, but recent data has shown that in fact this process is much more complicated than previously thought. Without wishing to be bound by any particular scientific theory, the first step in the digestion-absorption process of a typical vitamin E preparation after food ingestion is the dissolution of vitamin E into the lipid phase of the meal. The fat digestion process begins in the oral cavity by lingual lipase and continues in the stomach with the addition of gastric lipase produced by chief cells. There appears to be no metabolism (i.e., breakdown or absorption) of vitamin E in the stomach. The majority of fat digestion occurs in the duodenum by pancreatic lipase and colipase with the assistance of emulsifying bile acids. Lipids are emulsified into lipid droplets at both the gastric and duodenal levels. In addition, the size of the droplets does not appear to have any effect on the efficiency of subsequent absorption of vitamin E. In the duodenum, monoglycerides and long-chain fatty acids resulting from lipolysis form micelles with bile salts along with phospholipids, cholesterol, and fat-soluble vitamins. Vitamin E is incorporated into the mixed micelles along with lipid digestion products, a step towards absorption by enterocytes. The mixed micelles solubilize the hydrophobic components, allowing them to diffuse into the unstirred aqueous layer (glycocalyx) and gain access to the brush border membrane of the enterocyte. Once in close proximity, the mixed micelles may dissociate due to the existing pH gradient. The released components are then captured by various systems (e.g., passive diffusion and / or scavenger receptors) for absorption into the enterocyte. Once absorbed, the fate of vitamin E across the intestinal tract is not well described. Due to its hydrophobicity, vitamin E is likely to localize to organelle membranes, cytoplasmic lipid droplets, or traffic bound to binding proteins. Subcellular localization revealed that vitamin E can accumulate in microsomal membranes, i.e., endoplasmic reticulum, Golgi, lysosomal and peroxisomal membranes. Long-chain fatty acids and monoglycerides are re-esterified to triglycerides, which are subsequently combined with polypeptides, phospholipids and cholesterol to form chylomicrons. Most of vitamin E is incorporated into chylomicrons in its free form at the Golgi level and then released into the lymph.Chylomicrons then enter the intestinal lymphatics via the thoracic duct and eventually into the peripheral circulation. The lymphatic system circulates various immune system cells and is also the primary site of absorption of certain nutrients. Larger molecular weight digestive products are absorbed into the lymphatic system before reaching the systemic circulation via the venous system. Overall, the mechanism of vitamin E absorption is still only partially understood. Those skilled in the art will generally understand that the majority of vitamin E absorption occurs in the distal intestine.

[0160] Without wishing to be bound by any particular scientific theory, the inventors have surprisingly discovered that orally administered polypeptides in a core or suspension comprising a vitamin E agent and a polypeptide are effectively delivered to the bloodstream, for example, via the thoracic duct and / or lymphatic system.Thus, the present disclosure provides a general platform or system, including the compositions and methods disclosed herein, for delivering various polypeptides to the thoracic duct, lymphatic system, and / or bloodstream.

[0161] The present disclosure provides compositions and methods, for example, for delivery of a polypeptide to a subject, for example, delivery to the thoracic duct, lymphatic system, and / or bloodstream of a subject. In various embodiments, a composition or method of the present disclosure delivers a polypeptide to the bloodstream with beneficial pharmacokinetic properties. In various embodiments, the beneficial pharmacokinetic properties may result from distribution of the polypeptide through the lymphatic system (e.g., via the thoracic duct). Thus, the present disclosure specifically includes methods and compositions for delivery of a polypeptide to the lymphatic system (e.g., via the thoracic duct).

[0162] In various embodiments, the polypeptides of the polypeptide formulation are characterized by having a median, mean, or mode half-life of at least 15 hours across multiple samples, systems, and / or polypeptide molecules (e.g., across multiple subjects following administration of the polypeptide formulation to each subject), and optionally the half-life is at least 20, 25, 30, 35, 40, 45, 50, 55, 60 hours or more, and optionally the median, mean, or mode half-life is in a range having a lower limit of 30, 35, 40, 45, or 50 hours and an upper limit of 35, 40, 45, 50, 55, 60 hours or more. In various embodiments, following oral administration of a polypeptide formulation of the disclosure to a subject, the polypeptide of the polypeptide formulation has a half-life of at least 15 hours, and optionally the half-life is at least 20, 25, 30, 35, 40, 45, 50, 55, or 60 hours or more, and optionally the half-life is in a range having a lower limit of 30, 35, 40, 45, or 50 hours and an upper limit of 35, 40, 45, 50, 55, 60 hours or more.

[0163] In various embodiments, the polypeptide of the polypeptide formulation is characterized in having a median, mean, or mode half-life across multiple samples, systems, and / or polypeptide molecules (e.g., across multiple subjects following oral administration of the polypeptide formulation to each subject) that is at least 10% greater relative to a baseline than the median, mean, or mode half-life achieved by injection (e.g., subcutaneous administration) of the polypeptide (e.g., according to a standard therapeutic route of administration for the polypeptide); and optionally, the median, mean, or mode half-life (e.g., following oral administration) of the polypeptide of the polypeptide formulation is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, or 4-fold greater relative to a baseline than the median, mean, or mode half-life achieved by injection (e.g., subcutaneous administration) of the polypeptide (e.g., according to a standard therapeutic route of administration for the polypeptide). In various embodiments, following oral administration of a polypeptide formulation to a subject, the polypeptide of the polypeptide formulation has a half-life that is at least 10% greater relative to baseline than the half-life achieved by injection (e.g., subcutaneous administration) of the polypeptide (e.g., according to a standard therapeutic route of administration for the polypeptide), and optionally the half-life of the polypeptide of the polypeptide formulation following oral administration to a subject is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, or 4-fold greater relative to baseline than the half-life achieved by injection (e.g., subcutaneous administration) of the polypeptide (e.g., according to a standard therapeutic route of administration for the polypeptide).

[0164] In various embodiments, the polypeptide of the polypeptide formulation is administered in multiple samples, systems, and / or polypeptide molecules (e.g., in multiple subjects after oral administration of the polypeptide formulation to each subject), and is administered via injection (e.g., subcutaneous administration) of the polypeptide (e.g., following a standard therapeutic administration route for the polypeptide). max T at least 10% greater than the median, mean, or mode of the standard maxand optionally, a T of the polypeptide of the polypeptide formulation (e.g., after oral administration) max The median, mean, or mode of T is the T achieved by injection (e.g., subcutaneous administration) of the polypeptide (e.g., according to the standard therapeutic administration route of the polypeptide). max is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, or 4-fold, 5-fold, 10-fold, or 20-fold greater than the median, mean, or mode of the T, relative to the baseline. In various embodiments, following oral administration of the polypeptide formulation to a subject, the polypeptide of the polypeptide formulation exhibits a T, relative to the baseline, that is greater than the T achieved by injection (e.g., subcutaneous administration) of the polypeptide (e.g., according to a standard therapeutic route of administration for the polypeptide). max T at least 10% greater than the reference max and optionally a T of the polypeptide of the polypeptide formulation after oral administration to a subject. max is achieved by injection (e.g., subcutaneous administration) of the polypeptide (e.g., according to the standard therapeutic administration route for the polypeptide). max is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2x, 3x, or 4x, 5x, 10x, or 20x greater than the reference.

[0165] In various embodiments, the polypeptide of the polypeptide formulation is characterized by having a bioavailability across multiple samples, systems, and / or polypeptide molecules (e.g., across multiple subjects following oral administration of the polypeptide formulation to each subject) that is at least 1% greater relative to a baseline than the median, mean, or mode bioavailability achieved by injection (e.g., subcutaneous administration) of the polypeptide (e.g., according to a standard therapeutic route of administration for the polypeptide); and optionally, the median, mean, or mode bioavailability of the polypeptide of the polypeptide formulation (e.g., following oral administration) is at least 1%, 1.5%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, or 5-fold greater relative to a baseline than the bioavailability achieved by injection (e.g., subcutaneous administration) of the polypeptide (e.g., according to a standard therapeutic route of administration for the polypeptide). In various embodiments, following oral administration of a polypeptide formulation to a subject, the polypeptide of the polypeptide formulation has a bioavailability that is at least 1% greater relative to a baseline than the bioavailability achieved by injection (e.g., subcutaneous administration) of the polypeptide (e.g., according to a standard therapeutic route of administration for the polypeptide), and optionally, the bioavailability of the polypeptide of the polypeptide formulation following oral administration to a subject is at least 1%, 1.5%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, or 5-fold greater relative to a baseline than the bioavailability achieved by injection (e.g., subcutaneous administration) of the polypeptide (e.g., according to a standard therapeutic route of administration for the polypeptide). In various embodiments, the polypeptides of the polypeptide formulation are characterized by having a median, mean, or mode bioavailability of at least 1%, e.g., at least 1%, 1.5%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, across multiple samples, systems, and / or polypeptide molecules (e.g., across multiple subjects following oral administration of the polypeptide formulation to each subject).

[0166] In various embodiments, the polypeptide of the polypeptide formulation is administered in multiple samples, systems, and / or polypeptide molecules (e.g., in multiple subjects after each subject has been orally administered the polypeptide formulation), as determined by C-reactive protein synthesis (e.g., C-reactive protein synthesis) achieved by injection (e.g., subcutaneous administration) of the polypeptide (e.g., following a standard therapeutic administration route for the polypeptide). max C at least 10% smaller than the median, mean, or mode of the standard max and optionally, a C of the polypeptide of the polypeptide formulation (e.g., after oral administration) max The median, mean, or mode of C is the C achieved by injection (e.g., subcutaneous administration) of the polypeptide (e.g., according to the standard therapeutic administration route for the polypeptide). max is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less than the median, mean, or mode of the reference. In various embodiments, following oral administration of the polypeptide formulation to a subject, the polypeptide of the polypeptide formulation is at least 20% greater than the C achieved by injection (e.g., subcutaneous administration) of the polypeptide (e.g., according to a standard therapeutic route of administration for the polypeptide). max C is at least 10% smaller than the standard max and optionally a C of the polypeptide of the polypeptide formulation after oral administration to a subject. max is achieved by injection (e.g., subcutaneous administration) of the polypeptide (e.g., following the standard therapeutic administration route for the polypeptide). max is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less than the reference.

[0167] In various embodiments, the methods of the disclosure can include administering a composition of the disclosure to a subject in need thereof, the composition comprising a polypeptide for the treatment of a disease or condition from which the subject suffers.

[0168] Further disclosed herein is a method of treating a disease or disorder in a mammal, comprising administering to the mammal a composition of the present disclosure comprising a therapeutically effective amount of a polypeptide, wherein the formulation further comprises a pharma- ceutically acceptable viscosity-reducing agent, aggregation-reducing agent, or other additive as described above, and wherein the therapeutic formulation is effective for treating the disease or disorder. In some embodiments, the therapeutic formulation has improved stability compared to a reference formulation. In some embodiments, the excipient compound is essentially pure.

[0169] In some embodiments, the therapeutic use of the compositions of the present disclosure is directed to the treatment of cancer, e.g., breast cancer, gastric cancer, non-Hodgkin's lymphoma, urothelial carcinoma / solid tumor, metastatic colorectal cancer, non-squamous non-small cell lung cancer, metastatic breast cancer, Hodgkin's lymphoma, biliary tract cancer, acute myeloid leukemia, prostate cancer, multiple myeloma, solid tumor of bone, neuroblastoma, pancreatic cancer, acute myeloid leukemia, metastatic melanoma, metastatic squamous non-small cell carcinoma, anaplastic astrocytoma, brain tumor, glioblastoma, glioma, head and neck cancer, Merkel cell carcinoma, The present invention may include the treatment and / or detection of nasopharyngeal carcinoma, esophageal cancer, hepatocellular carcinoma, refractory neuroblastoma, osteosarcoma, peritoneal cancer, fallopian tube cancer, mesothelioma, metastatic melanoma, renal cell carcinoma, NR-LU-10 for cancer, lupus, chronic lymphocytic leukemia, soft tissue sarcoma, ovarian cancer, bladder cancer, esophageal cancer, gastric nasopharyngeal carcinoma, adrenocortical carcinoma, HER2 positive breast cancer, adenocarcinoma, granulomatosis with polyangiitis (GPA), microscopic polyangiitis, idiopathic pulmonary fibrosis, focal segmental glomerulosclerosis, prolactinoma, and combinations thereof.

[0170] In some embodiments, therapeutic uses of the compositions of the present disclosure may include the treatment and / or detection of autoimmune diseases, such as rheumatoid arthritis (RA), osteoarthritis, juvenile idiopathic arthritis (JIA), psoriatic arthritis (PsA), ankylosing spondylitis (AS), Crohn's disease (CD), ulcerative colitis (UC), psoriasis vulgaris (Ps), systemic lupus erythematosus, lupus nephritis, familial cold autoinflammatory syndrome (FCAS), Sjogren's syndrome, and combinations thereof.

[0171] In some embodiments, therapeutic uses of the compositions of the present disclosure may include the treatment and / or detection of another immune-related disease, such as leukopenia, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), thrombotic microangiopathy (TMA), inflammatory bowel disease, ulcerative colitis and transplant rejection, surgery-related life-threatening uncontrolled bleeding, and combinations thereof.

[0172] In some embodiments, therapeutic uses of the compositions of the present disclosure may include the treatment and / or detection of infectious diseases, such as Clostridium difficile infection, Respiratory Syncytial Virus (RSV) disease, Anthrax, Flu virus infection, Influenza virus infection, Hepatitis B virus infection, Rabies virus infection, Invasive Candida infection, Bacterial septic shock, HIV infection, Hospital-acquired pneumonia, Staphylococcal infection, STEC (Shiga-like toxin producing Escherichia coli or E. coli serotype O121) infection causing diarrhea and HUS (Hemolytic Uremic Syndrome), Cytomegalovirus, Botulism, Ebola virus, and combinations thereof.

[0173] In some embodiments, therapeutic uses of the compositions of the present disclosure may include the treatment and / or detection of cardiovascular disease, such as cardiac ischemic complications, percutaneous coronary intervention, acute myocardial infarction, pulmonary embolism, deep vein thrombosis, arterial thrombosis or embolism, occlusion of arteriovenous cannulae, thrombocytopenia associated with chronic immune (idiopathic) thrombocytopenic purpura (ITP), and combinations thereof.

[0174] In some embodiments, therapeutic uses of the compositions of the present disclosure may include the treatment and / or detection of ophthalmic disorders, such as age-related macular degeneration (AMD), macular edema, retinal vein occlusion (RVO), diabetic macular edema, neuromyelitis optica, and combinations thereof.

[0175] In some embodiments, therapeutic uses of the compositions of the present disclosure may include the treatment and / or detection of respiratory disorders, such as asthma, chronic idiopathic urticaria, acute bronchospasm or asthmatic conditions, chronic obstructive pulmonary disease, and combinations thereof.

[0176] In some embodiments, therapeutic uses of the compositions of the present disclosure may include the treatment and / or detection of metabolic disorders, such as hyperlipidemia, type 1 and type 2 diabetes, hypercholesterolemia, dyslipidemia, and combinations thereof.

[0177] In some embodiments, therapeutic uses of the compositions of the present disclosure may include the treatment and / or detection of genetic disorders such as hemophilia A and B, Prader-Willi syndrome, Turner syndrome, cryopyrin-associated periodic syndromes (CAPS), Muckle-Wells syndrome (MWS), X-linked hypophosphatemia, sickle cell crisis, and combinations thereof.

[0178] In some embodiments, therapeutic uses of the compositions of the present disclosure may include the treatment and / or detection of bone-related diseases, such as osteoporosis, aplastic anemia, and combinations thereof.

[0179] In some embodiments, therapeutic uses of the compositions of the present disclosure may include venom removal, treatment and / or detection of other disorders including Alzheimer's disease, back pain (sciatica), migraines, atopic dermatitis, Duchenne muscular dystrophy, liver fibrosis, cystic fibrosis, Pseudomonas aeruginosa infections, ventilator-associated pneumonia, and combinations thereof.

[0180] In various embodiments, the disclosure provides methods of producing the formulations of the disclosure, including, for example, (1) preparing a core comprising an amorphous or crystallized polypeptide composition and a pharma- ceutically acceptable carrier comprising a vitamin E agent, and (2) encapsulating the core in a pharma- ceutically acceptable capsule. In various embodiments, the disclosure provides methods of producing the formulations of the disclosure, including, for example, (1) crystallizing a polypeptide to produce an amorphous or crystallized polypeptide composition, (2) preparing a core comprising a crystallized or amorphous polypeptide composition and a pharma- ceutically acceptable carrier comprising a vitamin E agent, and (3) encapsulating the core in a pharma- ceutically acceptable capsule. EXAMPLES

[0181] The following examples demonstrate methods and compositions for orally administering polypeptides, including polypeptides and vitamin E agents. The following examples further demonstrate the advantages of the oral formulations of the present disclosure, including improved half-life and bioavailability, as compared to a baseline liquid formulation of the same polypeptide for administration by injection.

[0182] This example provides for the formulation and testing of oral formulations comprising a polypeptide that is a crystallized or amorphous polypeptide and a vitamin E agent. This example utilizes batch crystallization to produce a crystallized polypeptide composition. As those skilled in the art will understand from this disclosure, the specific production method, size, size distribution, shape, and shape distribution of crystals are not essential characteristics for successfully formulating a polypeptide according to the methods and compositions provided herein. This example clearly demonstrates that the oral formulations of the present disclosure comprising amorphous or crystallized polypeptides can be advantageously produced.

[0183] Example 1: Crystallization of Trastuzumab This example illustrates the formation of crystallized trastuzumab for use in the oral formulation disclosed herein. Trastuzumab is a humanized monoclonal antibody commercially available as Herclon® (Roche). Trastuzumab is an IgG1 kappa antibody that contains human framework regions and the complementarity determining regions of a murine antibody (4D5) that binds to HER2. Trastuzumab is widely used to treat breast cancers that overexpress the extracellular domain of the epidermal growth factor receptor 2 (HER2) protein. This example includes the recognition that trastuzumab is exemplary of a polypeptide for which an oral formulation may be advantageous.

[0184] Trastuzumab was stored as a sterile lyophilized powder in its original 440 mg vial and subsequently dissolved in 5 ml of sterile water. The dissolved trastuzumab solution contained 88 mg / ml trastuzumab, 39.6 mg L-histidine HCl, 25.6 mg L-histidine, 1600 mg α,α-trehalose dihydrate, and 7.2 mg polysorbate 20 (USP).

[0185] To generate trastuzumab microparticles, a 500 μl aliquot of trastuzumab (88 mg / ml) in a buffer containing 3.96 mg / ml L-histidine hydrochloride, 2.56 mg / ml L-histidine, 160 mg / ml α,α-trehalose dihydrate, and 0.72 mg / ml polysorbate 20 (USP) was mixed with 1,000 μl of a reagent containing 20% ​​PEG300, 10% PEG8000, 10% glycerol, 100 mM Tris (pH 8.5) and incubated overnight at room temperature. The final concentration of trastuzumab in the solution was 29.33 mg / ml. The mixture was then mixed using a vortex and left at room temperature. The following day, trastuzumab microparticles were obtained (Figure 1). This method resulted in 95% of the input trastuzumab forming microparticles.

[0186] Example 2: Crystallization of Liraglutide This example illustrates the formation of crystallized liraglutide for use in oral formulations disclosed herein. Liraglutide is sold under the trade name Victoza® (Novo Nordisk). Liraglutide is a synthetic analog of human glucagon-like peptide-1 (GLP-1) produced using recombinant technology and acts as a GLP-1 receptor agonist. Liraglutide is 97% homologous to native human GLP-1, with arginine replacing lysine at position 34. Liraglutide contains a C-16 fatty acid (palmitic acid) with a glutamic acid spacer on the remaining lysine residue at position 26 of the peptide precursor. Therapeutic effects of liraglutide include increased insulin release from the pancreas and reduced excess glucagon release. This example includes the recognition that liraglutide is exemplary of a polypeptide for which oral formulation may be advantageous.

[0187] The half-life of liraglutide is an important consideration in its therapeutic use. Endogenous GLP-1 has a plasma half-life of 1.5-2 minutes due to degradation by the ubiquitous enzymes dipeptidyl peptidase-4 (DPP4) and neutral endopeptidase (NEP). The half-life after intramuscular injection is approximately 30 minutes, limiting its therapeutic use when administered this way. The metabolically active form of GLP-1 is endogenous GLP-1-(7-36)NH 2 , and the rarer GLP-1-(7-37). The sustained action of liraglutide is achieved by attaching a fatty acid molecule to a single position on the GLP-1-(7-37) molecule, allowing it to both self-associate and bind to albumin. Active GLP-1 is then released from albumin at a slow, steady rate.

[0188] For use in this example, liraglutide was obtained as VICTOZA, a clear or nearly colorless solution. Each mL of VICTOZA solution contains 6 mg of liraglutide and the following inactive ingredients: 1.42 mg disodium phosphate dihydrate, 14 mg propylene glycol, 5.5 mg phenol, and water for injection. Each prefilled pen contains 3 mL of solution of VICTOZA, equivalent to 18 mg of liraglutide (free base, anhydrous).

[0189] To generate liraglutide microparticles, liraglutide was dialyzed against three changes of water for 24 hours and lyophilized to obtain a dry powder. The lyophilized powder of liraglutide was then dissolved in water at a concentration of 80 mg / mL. Aliquots of liraglutide were then crystallized using various methods described below.

[0190] Crystallization method 1: An aliquot of 500 μl of liraglutide (80 mg / ml) in water was mixed with 1,000 μl of a reagent containing 1.34 M ammonium sulfate, 3.35% (v / v) PEG400, 0.05 M magnesium sulfate, and 0.1 M Tris base / HCl pH 8.5 and incubated overnight at room temperature. The final concentration of liraglutide in the solution was 26.67 mg / ml. The mixture was then mixed using a vortex and left at room temperature. Liraglutide microparticles were obtained the next day (Figure 2). This method resulted in 75% of the input liraglutide forming microparticles.

[0191] Crystallization method 2: An aliquot of 500 μl liraglutide (80 mg / ml) in water was mixed with 1,000 μl of a reagent containing 35% (v / v) MPD, 100 mM Tris base / HCl pH 7.0, 200 mM sodium chloride, and incubated overnight at room temperature. The final concentration of liraglutide in the solution was 26.67 mg / ml. The mixture was then mixed using a vortex and left at room temperature. Liraglutide microparticles were obtained the next day (Figure 3). With this method, 85% of the input liraglutide formed microparticles.

[0192] Crystallization method 3: An aliquot of 500 μl liraglutide (80 mg / ml) in water was mixed with 1,000 μl of a reagent containing 1,000 mM dibasic ammonium phosphate, 100 mM sodium citrate / citric acid pH 5.5, 200 mM sodium chloride, and incubated overnight at room temperature. The final concentration of liraglutide in the solution was 26.67 mg / ml. The mixture was then mixed using a vortex and left at room temperature. Liraglutide microparticles were obtained the next day (Figure 4). This method resulted in 90% of the input liraglutide forming microparticles.

[0193] Crystallization method 4: An aliquot of 500 μl liraglutide (80 mg / ml) in water was mixed with 1,000 μl of a reagent containing 25% (w / v) PEG1500, 100 mM SPG buffer pH 8.5 and incubated overnight at room temperature. The final concentration of liraglutide in the solution was 26.67 mg / ml. The mixture was then mixed using a vortex and left at room temperature. The next day, liraglutide microparticles were obtained (Figure 5). With this method, 85% of the input liraglutide formed microparticles.

[0194] Crystallization method 5: An aliquot of 500 μl liraglutide (80 mg / ml) in water was mixed with 1,000 μl of a reagent containing 40% (v / v) ethylene glycol, 100 mM MES / sodium hydroxide pH 6.0, 200 mM zinc acetate, and incubated overnight at room temperature. The final concentration of liraglutide in the solution was 26.67 mg / ml. The mixture was then mixed using a vortex and left at room temperature. Liraglutide microparticles were obtained the next day (Figure 6). This method resulted in 75% of the input liraglutide forming microparticles.

[0195] Crystallization method 6: An aliquot of 500 μl liraglutide (80 mg / ml) in water was mixed with 1,000 μl of a reagent containing 3 M sodium chloride, 5% (v / v) MPD, 0.1 M calcium chloride, 0.1 M imidazole / HCl pH 6.5 and incubated overnight at room temperature. The final concentration of liraglutide in the solution was 26.67 mg / ml. The mixture was then mixed using a vortex and left at room temperature. Liraglutide microparticles were obtained the next day (Figure 7). With this method, 80% of the input liraglutide formed microparticles.

[0196] Crystallization method 7: An aliquot of 500 μl liraglutide (80 mg / ml) in water was mixed with 1,000 μl of a reagent containing 0.429 M sodium chloride, 9.9% (v / v) isopropanol, 0.1 M calcium chloride, 0.1 M imidazole / hydrochloric acid pH 6.5 and incubated overnight at room temperature. The final concentration of liraglutide in the solution was 26.67 mg / ml. The mixture was then mixed using a vortex and left at room temperature. Liraglutide microparticles were obtained the next day (Figure 8). This method resulted in 70% of the input liraglutide forming microparticles.

[0197] Crystallization method 8: An aliquot of 500 μl liraglutide (80 mg / ml) in water was mixed with 1,000 μl of a reagent containing 5.36% (v / v) MPD, 0.67 M potassium phosphate dibasic / sodium phosphate monobasic pH 8.5 and incubated overnight at room temperature. The final concentration of liraglutide in the solution was 26.67 mg / ml. The mixture was then mixed using a vortex and left at room temperature. The next day, liraglutide microparticles were obtained (Figure 9). This method resulted in 85% of the input liraglutide forming microparticles.

[0198] Example 3: Crystallization of insulin This example illustrates the formation of crystalline insulin for use in an oral formulation as disclosed herein. Humulin® R is a polypeptide hormone structurally identical to human insulin, synthesized through rDNA technology in a non-disease producing laboratory strain of Escherichia coli bacteria. Humulin R is indicated as an adjunct treatment to diet and exercise to improve glycemic control in adult and pediatric patients with type 1 and type 2 diabetes. When used subcutaneously, Humulin R is typically administered three or more times daily before meals. This example includes the recognition that insulin is exemplary of a polypeptide for which an oral formulation may be advantageous.

[0199] This example utilizes Humulin R (Insulin (Human Recombinant)) U-100 (100 units / mL). To generate insulin microparticles, insulin (Sigma Chemical Company) was desalted with 0.01N NaOH as follows: 62 mg of insulin was dissolved in 4 mL of 0.01N HCl (pH 2.0). The pH of the polypeptide solution was increased until the polypeptide precipitated by adding 10 uL of 0.1N NaOH each time. The precipitated polypeptide was collected and washed twice with 5 mL of water, collecting the water separately each time. The dissolved polypeptide was collected from the water by adding 0.1N NaOH to the collected water. Finally, all the precipitate was dissolved in 1 mL of 0.01N HCL. Aliquots of insulin were then crystallized using various methods described below.

[0200] Crystallization method 1: An aliquot of 500 μl insulin (approximately 62 mg / ml) in water was mixed with 1,000 μl of a reagent containing 40% (v / v) PEG300, 100 mM sodium cacodylate / HCl pH 6.5, 200 mM calcium acetate, and incubated overnight at room temperature. The final concentration of insulin in the solution was 15.5 mg / ml. The mixture was then mixed using a vortex and left at room temperature. Insulin microparticles were obtained the next day (Figure 10). With this method, 90% of the insulin input formed microparticles.

[0201] Crystallization method 2: An aliquot of 500 μl insulin (approximately 62 mg / ml) in water was mixed with 1,000 μl of a reagent containing 40% (v / v) PEG600, 100 mM sodium cacodylate / HCl pH 6.5, 200 mM calcium acetate, and incubated overnight at room temperature. The final concentration of insulin in the solution was 15.5 mg / ml. The mixture was then mixed using a vortex and left at room temperature. Insulin microparticles were obtained the next day (Figure 11). With this method, 90% of the insulin input formed microparticles.

[0202] Crystallization method 3: An aliquot of 500 μl insulin (approximately 62 mg / ml) in water was mixed with 1,000 μl of a reagent containing 40% (v / v) 1,2-propanediol, 100 mM sodium acetate / acetic acid pH 4.5, 50 mM calcium acetate, and incubated overnight at room temperature. The final concentration of insulin in the solution was 15.5 mg / ml. The mixture was then mixed using a vortex and left at room temperature. Insulin microparticles were obtained the next day (Figure 12). With this method, 90% of the insulin input formed microparticles.

[0203] Crystallization method 4: An aliquot of 500 μl insulin (approximately 62 mg / ml) in water was mixed with 1,000 μl of a reagent containing 14.4% (w / v) PEG8000, 80 mM sodium cacodylate / HCl pH 6.5, 160 mM calcium acetate, 20% (v / v) glycerol and incubated overnight at room temperature. The final concentration of insulin in the solution was 15.5 mg / ml. The mixture was then mixed using a vortex and left at room temperature. Insulin microparticles were obtained the next day (Figure 13). With this method, 90% of the insulin input formed microparticles.

[0204] Crystallization method 5: An aliquot of 500 μl insulin (approximately 62 mg / ml) in water was mixed with 1,000 μl of a reagent containing 20% ​​(w / v) PEG3000, 100 mM Tris base / HCl pH 7.0, 200 mM calcium acetate, and incubated overnight at room temperature. The final concentration of insulin in the solution was 15.5 mg / ml. The mixture was then mixed using a vortex and left at room temperature. Insulin microparticles were obtained the next day (Figure 14). With this method, 90% of the insulin input formed microparticles.

[0205] Crystallization method 6: An aliquot of 500 μl insulin (approximately 62 mg / ml) in water was mixed with 1,000 μl of a reagent containing 20% ​​(w / v) PEG1000, 100 mM sodium cacodylate / hydrochloric acid pH 6.5, 200 mM magnesium chloride, and incubated overnight at room temperature. The final concentration of insulin in the solution was 15.5 mg / ml. The mixture was then mixed using a vortex and left at room temperature. Insulin microparticles were obtained the next day (Figure 15). With this method, 90% of the insulin input formed microparticles.

[0206] Crystallization method 7: An aliquot of 500 μl insulin (approximately 62 mg / ml) in water was mixed with 1,000 μl of a reagent containing 10% (v / v) 2-propanol, 100 mM MES / sodium hydroxide pH 6.0, 200 mM calcium acetate, and incubated overnight at room temperature. The final concentration of insulin in the solution was 15.5 mg / ml. The mixture was then mixed using a vortex and left at room temperature. Insulin microparticles were obtained the next day (Figure 16). With this method, 90% of the insulin input formed microparticles.

[0207] Example 4: Pharmacokinetic study of a liraglutide formulation containing vitamin E in New Zealand White rabbits This example demonstrates that an oral formulation of liraglutide, exemplary of the oral formulations disclosed herein, has a therapeutically effective pharmacokinetic profile with advantageous properties compared, for example, to parenteral administration of the same polypeptide.

[0208] This example includes an oral formulation of crystallized polypeptide prepared as follows: Commercially available Victoza® (Novo Nordisk) (3 mL solution contains 6 mg / mL liraglutide, 5.5 mg phenol, sodium hydroxide, hydrochloric acid, and water for injection) was dialyzed against water for 24 hours at 4° C. with three changes. The dialyzed liraglutide was then used to prepare microparticles according to the procedure mentioned in Example 2. The microparticle liraglutide was then washed with cold isopropanol before being freeze-dried. The freeze-dried liraglutide (dosage mentioned in Table 2) was then transferred into a size 1 clear gelatin capsule containing 50 μL vitamin E (Sigma Chemical Company). The capsule was then stored at 4° C. until further use.

[0209] Pharmacokinetic analysis was performed in male New Zealand White rabbits weighing 1.5 kg for 5 days. The experimental design is shown in Table 2. Rabbits were fed with sodium bicarbonate-containing diet (20 gm normal diet + 3 gm sodium bicarbonate (pellets / feed balls prepared by adding water)). Animals were fed with the above diet from day -3 to day 0 before dosing and then fed with normal diet only during the experimental period. Plasma samples taken at different time points were analyzed by LRT ELISA kit according to ABBEXA's protocol. The kit is based on competitive enzyme-linked immunosorbent assay technology. Antibody anti-liraglutide was pre-coated on a 96-well plate. Standards, test samples and biotin-conjugated reagent were added to the wells and incubated. A competitive inhibition reaction occurs between biotin-labeled LRT and unlabeled LRT on the pre-coated antibody. HRP-conjugated reagent was then added and the whole plate was incubated. At each step, unbound conjugates were removed with washing buffer. HRP enzyme reaction was quantified using TMB substrate. After adding TMB substrate, only wells with enough LRT will produce a blue product, which then turns yellow after adding an acidic stop solution. The intensity of the yellow color is inversely proportional to the amount of LRT bound on the plate. The OD at 450 nm is measured spectrophotometrically in a microplate reader, from which the concentration of LRT can be calculated.

[0210] Liraglutide was analyzed in plasma using a specific enzyme-linked immunosorbent assay (ELISA). Pharmacokinetic endpoints were determined using noncompartmental methods with PK Solutions software. AUC was approximated using the trapezoidal rule for observed concentrations. C max is derived as the maximum of all effective concentrations, and then t maxを C max The terminal elimination rate constant (λ z ) was estimated by log-linear regression on the terminal portion of the concentration-time curve, and then t 1 / 2 A 1 / 2 = ln2 / λ z It was calculated as:

[0211] The data in Table 3 and Figure 17 show the mean plasma concentrations of liraglutide at various time points. The orally administered liraglutide formulation showed 39% bioavailability compared to the subcutaneous formulation. [Table 2] [Table 3]

[0212] Example 5: Pharmacokinetic study of liraglutide formulation containing vitamin E in beagle dogs This example demonstrates that an oral formulation of liraglutide, exemplary of the oral formulations disclosed herein, has a therapeutically effective pharmacokinetic profile with advantageous properties compared, for example, to parenteral administration of the same polypeptide.

[0213] This example includes an oral formulation of crystallized polypeptide prepared as follows: Commercially available Victoza® (Novo Nordisk) (3 mL solution contains 6 mg / mL liraglutide, 5.5 mg phenol, sodium hydroxide, hydrochloric acid, and water for injection) was dialyzed against water for 24 hours at 4° C. with three changes. The dialyzed liraglutide was then treated according to the procedure mentioned in Example 2 to prepare microparticles. The microparticle liraglutide was then washed with cold isopropanol before being freeze-dried. The freeze-dried liraglutide (dosage mentioned in Table 4) was then transferred into a size 1 clear gelatin capsule containing 50 μL vitamin E (Sigma Chemical Company). The capsule was then stored at 4° C. until further use.

[0214] Pharmacokinetic analysis was performed for 5 days using beagle dogs weighing 15 kg. The experimental design is shown in Table 4. Plasma samples taken at different time points were analyzed by LRT ELISA kit according to the ABBEXA protocol. The kit is based on competitive enzyme-linked immunosorbent assay technology. Antibody anti-liraglutide was pre-coated on a 96-well plate. Standards, test samples, and biotin-conjugated reagent were added to the wells and incubated. A competitive inhibition reaction occurs between biotin-labeled LRT and unlabeled LRT on the pre-coated antibody. HRP-conjugated reagent was then added and the whole plate was incubated. At each stage, unbound conjugates were removed with washing buffer. TMB substrate was used to quantify the HRP enzyme reaction. After adding TMB substrate, only wells with sufficient LRT produced a blue product, which then turned yellow after adding an acidic stop solution. The intensity of the yellow color is inversely proportional to the amount of LRT bound on the plate. The OD at 450 nm was measured spectrophotometrically on a microplate reader, from which the concentration of LRT can be calculated.

[0215] The data in Table 5 and Figure 18 show the mean plasma concentrations of liraglutide at various time points. The orally administered liraglutide formulation (15 mg in capsule formulation) showed 19% bioavailability compared to the subcutaneous control (subcutaneous, 1.5 mg, second injection 96 hours after first injection).

[0216] Liraglutide was analyzed in plasma using a specific enzyme-linked immunosorbent assay (ELISA). Pharmacokinetic endpoints were determined using noncompartmental methods with PK Solutions software. AUC was approximated using the trapezoidal rule for observed concentrations. C max is derived as the maximum of all effective concentrations, and then t maxを C max The terminal elimination rate constant (λ z ) was estimated by log-linear regression on the terminal portion of the concentration-time curve, and then t 1 / 2 A 1 / 2 = ln2 / λ z It was calculated as: [Table 4] [Table 5]

[0217] Example 6: Pharmacokinetic study of a liraglutide formulation containing vitamin E in minipigs This example demonstrates that an oral formulation of liraglutide, exemplary of the oral formulations disclosed herein, has a therapeutically effective pharmacokinetic profile with advantageous properties compared, for example, to parenteral administration of the same polypeptide.

[0218] This example includes an oral formulation of crystallized polypeptide prepared as follows: Commercially available Victoza® (Novo Nordisk) (3 mL solution contains 6 mg / mL liraglutide, 5.5 mg phenol, sodium hydroxide, hydrochloric acid, and water for injection) was dialyzed against water for 24 hours at 4° C. with three changes. The dialyzed liraglutide was then processed according to the procedure mentioned in Example 2 to prepare microparticles. The microparticle liraglutide was then washed with cold isopropanol before being freeze-dried. The freeze-dried liraglutide (dosage mentioned in Table 6) was then transferred into a size 1 clear gelatin capsule containing 50 μL vitamin E (Sigma Chemical Company). The capsule was then stored at 4° C. until further use.

[0219] Pharmacokinetic analysis was performed for 5 days using minipigs weighing 12 kg. The experimental design is shown in Table 6. Plasma samples taken at different time points were analyzed by LRT ELISA kit according to the ABBEXA protocol. The kit is based on competitive enzyme-linked immunosorbent assay technology. Antibody anti-liraglutide was pre-coated on a 96-well plate. Standards, test samples, and biotin-conjugated reagent were added to the wells and incubated. A competitive inhibition reaction occurs between biotin-labeled LRT and unlabeled LRT on the pre-coated antibody. HRP-conjugated reagent was then added and the whole plate was incubated. At each stage, unbound conjugates were removed with washing buffer. TMB substrate was used to quantify the HRP enzyme reaction. After adding TMB substrate, only wells with sufficient LRT produced a blue product, which then turned yellow after adding an acidic stop solution. The intensity of the yellow color is inversely proportional to the amount of LRT bound on the plate. The OD at 450 nm was measured spectrophotometrically in a microplate reader, from which the concentration of LRT can be calculated.

[0220] The data in Table 7 and Figure 19 show the mean plasma concentrations of liraglutide at various time points. The orally administered liraglutide formulation (1.250 mg in capsule formulation) showed 38% bioavailability compared to subcutaneous (0.0625 mg).

[0221] Liraglutide was analyzed in plasma using a specific enzyme-linked immunosorbent assay (ELISA). Pharmacokinetic endpoints were determined using noncompartmental methods with PK Solutions software. AUC was approximated using the trapezoidal rule for observed concentrations. C max is derived as the maximum of all effective concentrations, and then t maxを C max The terminal elimination rate constant (λ z ) was estimated by log-linear regression on the terminal portion of the concentration-time curve, and then t 1 / 2 A 1 / 2 = ln2 / λ z It was calculated as: [Table 6] [Table 7]

[0222] Example 7: Pharmacokinetic study of a vitamin E-containing trastuzumab formulation in New Zealand White rabbits This example demonstrates that oral formulations of trastuzumab, exemplary of the oral formulations disclosed herein, have a therapeutically effective pharmacokinetic profile with advantageous properties compared, for example, to parenteral administration of the same polypeptide.

[0223] Oral formulations of crystallized polypeptides included in this example were prepared as follows: Commercially obtained trastuzumab, HERCEPTIN® (lyophilized powder containing 440 mg trastuzumab, 9.9 mg L-histidine HCl, 6.4 mg L-histidine, 400 mg α,α,-trehalose dihydrate, and 1.8 mg polysorbate 20 (USP), and 20 mL water as water for reconstitution injection), was reconstituted in 6 mL water for injection. The reconstituted trastuzumab was then processed according to the procedure mentioned in Example 1 to prepare microparticles. The microparticle trastuzumab was then washed with cold isopropanol. Trastuzumab (dosages mentioned in Table 8) was then transferred into size 00 clear gelatin capsules containing 50 μL vitamin E (Sigma Chemical Company). The capsules were then stored at 4° C. until further use.

[0224] Pharmacokinetic analysis was performed in male New Zealand White rabbits weighing 1.5 kg for 14 days. The experimental design is shown in Table 8. Rabbits were fed with sodium bicarbonate-containing diet (20 gm normal diet + 3 gm sodium bicarbonate (pellets / feed balls prepared by adding water)). Animals were fed the above diet from day -3 to day 0 before dosing, and then fed with normal diet only during the experimental period. Plasma samples taken at different time points were analyzed by ELISA. Briefly, MAXISORP NUNC immunoplates were coated with 2 μg / mL anti-trastuzumab coating antibody (Abcam) in sodium carbonate buffer pH 9.6 overnight at 4°C. The coated plates were washed with washing buffer (PBS + 0.01% Tween® 20). After washing, the plates were blocked with blocking buffer (PBS + 1% BSA) for 3 hours at room temperature. Plasma samples to be analyzed were diluted with PBS and incubated with the coating antibody for 1 h, then washed and a goat anti-human IgG (Fc specific)-peroxidase capture antibody (Sigma) was added. Detection was performed at 450 nm using TMB substrate. result The data in Table 9 and Figure 20 show the mean plasma concentrations of trastuzumab at various time points. The orally administered trastuzumab formulation showed 26% bioavailability compared to the intravenous administration (reconstituted Herclon (Roche) vial contains 21 mg / mL trastuzumab, L-histidine hydrochloride, L-histidine, -trehalose dihydrate, polysorbate 20, and water for injection). Trastuzumab was analyzed in plasma using a specific enzyme-linked immunosorbent assay (ELISA). Pharmacokinetic endpoints were determined using noncompartmental methods with PK Solutions software. AUCs were approximated using the trapezoidal rule for observed concentrations. C max is derived as the maximum of all effective concentrations, and then t maxを C max The terminal elimination rate constant (λ z) was estimated by log-linear regression on the terminal portion of the concentration-time curve, and then t 1 / 2 A 1 / 2 = ln2 / λ z It was calculated as: [Table 8] [Table 9]

[0225] Example 8: Pharmacokinetic study of a vitamin E-containing trastuzumab formulation in New Zealand White rabbits This example demonstrates that oral formulations of trastuzumab, exemplary of the oral formulations disclosed herein, have a therapeutically effective pharmacokinetic profile with advantageous properties compared, for example, to parenteral administration of the same polypeptide.

[0226] Oral formulations of crystallized polypeptides included in this example were prepared as follows: Commercially obtained trastuzumab, HERCEPTIN® (lyophilized powder containing 440 mg trastuzumab, 9.9 mg L-histidine HCl, 6.4 mg L-histidine, 400 mg α,α,-trehalose dihydrate, and 1.8 mg polysorbate 20 (USP), and 20 mL water as water for reconstitution injection), was reconstituted in 6 mL water for injection. The reconstituted trastuzumab was then treated according to the procedure mentioned in Example 1 to prepare microparticles. The microparticle trastuzumab was then washed with cold isopropanol before being lyophilized. The lyophilized trastuzumab (dosages mentioned in Table 10) was then transferred into size 00 clear gelatin capsules containing 50 μL vitamin E (Sigma Chemical Company). The capsules were then stored at 4° C. until further use.

[0227] Pharmacokinetic analysis was performed in male New Zealand White rabbits weighing 1.5 kg for 5 days. The experimental design is shown in Table 10. Rabbits were fed with sodium bicarbonate-containing diet (20 gm normal diet + 3 gm sodium bicarbonate (pellets / feed balls prepared by adding water)). Animals were fed the above diet from day -3 to day 0 before dosing, and then fed with normal diet only during the experimental period. Plasma samples taken at different time points were analyzed by ELISA. Briefly, MAXISORP NUNC immunoplates were coated with 2 μg / mL anti-trastuzumab coating antibody (Abcam) in sodium carbonate buffer pH 9.6 overnight at 4°C. The coated plates were washed with washing buffer (PBS + 0.01% Tween® 20). After washing, the plates were blocked with blocking buffer (PBS + 1% BSA) for 3 hours at room temperature. Plasma samples to be analyzed were diluted with PBS and incubated with the coating antibody for 1 h, then washed and a goat anti-human IgG (Fc specific)-peroxidase capture antibody (Sigma) was added. Detection was performed at 450 nm using TMB substrate.

[0228] The data in Table 11 and Figure 21 show the mean plasma concentrations of trastuzumab at various time points. The orally administered trastuzumab formulation (Herclon (Roche) reconstituted vial contains 21 mg / mL trastuzumab, L-histidine hydrochloride, L-histidine, trehalose dihydrate, polysorbate 20, and water for injection) showed 2% bioavailability compared to the intravenous administration. Trastuzumab was analyzed in plasma using a specific enzyme-linked immunosorbent assay (ELISA). Pharmacokinetic endpoints were determined using noncompartmental methods using PK Solutions software. AUC was approximated using the trapezoidal rule for the observed concentrations. C max is derived as the maximum of all effective concentrations, and then t max C max The terminal elimination rate constant (λ z ) was estimated by log-linear regression on the terminal portion of the concentration-time curve, and then t1 / 2 A 1 / 2 = ln2 / λ z It was calculated as: [Table 10] [Table 11]

[0229] Example 9: Pharmacokinetic study of insulin formulations containing vitamin E in New Zealand White rabbits This example demonstrates that oral formulations of insulin, exemplary of the oral formulations disclosed herein, have a therapeutically effective pharmacokinetic profile with advantageous properties compared to, for example, parenteral administration of the same polypeptide. This example demonstrates insulin suspension formulations of vitamin E and demonstrates that enteric formulations are not required for advantageous application of the formulations disclosed herein.

[0230] This example includes an oral formulation of crystallized polypeptide prepared as follows: Commercially obtained regular insulin, Humulin R (insulin human recombinant) U-100, is a sterile, clear, aqueous, and colorless solution containing 100 units / mL human insulin (rDNA derived), 16 mg / mL glycerin, and 2.5 mg / mL meta-cresol, endogenous zinc (approximately 0.015 mg / 100 units), and water for injection. The pH is 7.0-7.8. The insulin was dialyzed against water for 24 hours and lyophilized. The lyophilized insulin was reconstituted with dilute acid. The reconstituted insulin was then treated according to the procedure mentioned in Example 3 to prepare microparticles. The microparticle insulin was then washed with cold isopropanol and then lyophilized. The lyophilized insulin (dosage mentioned in Table 12) was then suspended in 500 μL of vitamin E (Sigma Chemical Company). The sample was then stored at 4° C. until further use.

[0231] Pharmacokinetic analysis was performed in male New Zealand White rabbits weighing 1.5 kg for 2 days. The experimental design is shown in Table 12. Rabbits were fed with sodium bicarbonate-containing diet (20 gm normal diet + 3 gm sodium bicarbonate (pellets / feed balls prepared by adding water)). Animals were fed the above diet from day -3 to day 0 before dosing, and then fed with normal diet only during the experimental period. Oral delivery of insulin was performed through a G-tube reaching the stomach. The G-tube was then flushed with 10 mL coconut oil to remove the sample adhering to the tube wall. Plasma samples taken at different time points were analyzed by ELISA. Briefly, MAXISORP NUNC immunoplates were coated with 2 μg / mL anti-insulin coating antibody (Crystal Chem) in sodium carbonate buffer pH 9.6 overnight at 4° C. The coated plates were washed with washing buffer (PBS + 0.01% Tween® 20). After washing, the plates were blocked with blocking buffer (PBS + 1% BSA) for 3 h at room temperature. Plasma samples to be analyzed were diluted in PBS and incubated with the coating antibody for 1 h, then washed and a goat anti-human IgG (Fc specific)-peroxidase capture antibody (Sigma) was added. Detection was performed at 450 nm using TMB substrate.

[0232] The data in Table 13 and Figure 22 show the mean plasma concentrations of insulin at various time points. The orally administered insulin formulation showed 4% bioavailability compared to the intravenous administration. Insulin was analyzed in plasma using a specific enzyme-linked immunosorbent assay (ELISA). Pharmacokinetic endpoints were determined using non-compartmental methods using PK Solutions software. AUC was approximated using the trapezoidal rule for the observed concentrations. C for each insulin dose was 0.01 mg / kg / day. max is derived as the maximum of all effective concentrations, and then t maxを C max The terminal elimination rate constant (λ z ) was estimated by log-linear regression on the terminal portion of the concentration-time curve, and then t 1 / 2 A1 / 2 = ln2 / λ z It was calculated as: [Table 12] [Table 13]

[0233] Example 10: Pharmacokinetic study of trastuzumab formulations in New Zealand White rabbits This example demonstrates that oral formulations of trastuzumab formulated with vitamin E have unique properties for oral administration compared to formulations with other similar pharma- ceutical acceptable carriers. This example demonstrates a suspension formulation of trastuzumab in vitamin E and demonstrates that an enteric formulation is not required for advantageous application of the formulations of the present disclosure.

[0234] Oral formulations of the crystallized polypeptides included in this example were prepared as follows: Commercially obtained trastuzumab, HERCEPTIN® (lyophilized powder containing 440 mg trastuzumab, 9.9 mg L-histidine HCl, 6.4 mg L-histidine, 400 mg α,α,-trehalose dihydrate, and 1.8 mg polysorbate 20, USP, with 20 mL water as water for reconstitution), was reconstituted in 6 mL water for injection. The reconstituted trastuzumab was then processed as described in the above example to prepare microparticles. The microparticle trastuzumab was then washed with cold isopropanol. Samples of microparticle trastuzumab were suspended at 21 mg / mL in each of the following pharma-ceutically acceptable carriers for oral formulations: vitamin E, coconut oil, cod liver oil, and ghee.

[0235] Pharmacokinetic analysis was performed in male New Zealand White rabbits weighing 1.5 kg for 14 days. The experimental design is shown in Table 14. Each oral formulation was orally administered as shown in Table 14. A control standard of care formulation (containing neither microparticles nor suspension) was also administered by subcutaneous injection as shown in Table 14 (a reconstituted Herclon (Roche) vial contains 21 mg / mL trastuzumab, L-histidine hydrochloride, L-histidine, α,α,-trehalose dihydrate, polysorbate 20 and water for injection). The rabbits were fed a diet containing sodium bicarbonate (20 gm of normal chow + 3 gm of sodium bicarbonate (pellets / feed balls prepared by adding water)). The animals were fed the above diet from day -3 to day 0 before dosing, and then fed the normal chow only during the experimental period. Oral dosing to the animals was performed using a G tube. The suspension formulation was passed through the G tube using a syringe. After dosing, the tubes were washed with 5 mL of coconut oil followed by 5 mL of water.

[0236] Plasma samples taken at different time points were analyzed by ELISA. Briefly, MAXISORP NUNC immunoplates were coated with 2 μg / mL anti-trastuzumab coating antibody (Abcam) in sodium carbonate buffer pH 9.6 overnight at 4° C. The coated plates were washed with washing buffer (PBS+0.01% Tween® 20). After washing, the plates were blocked with blocking buffer (PBS+1% BSA) for 3 hours at room temperature. Plasma samples to be analyzed were diluted in PBS and incubated with the coating antibody for 1 hour, then washed and goat anti-human IgG (Fc specific)-peroxidase capture antibody (Sigma) was added. Detection was performed at 450 nm using TMB substrate. [Table 14]

[0237] result The data in Figure 23 shows the mean plasma concentration of insulin at various time points. Coconut oil, ghee, or cod liver oil did not show significant plasma concentrations above zero at any time point, whereas the subcutaneous administration of the commercial "Innovator" formulation and the oral vitamin E formulation both produced significant plasma concentrations (e.g., about 50 μg / mL at 100 hours for the oral vitamin E formulation and about 150 μg / mL at 100 hours for the subcutaneous administration of the commercial "Innovator" formulation). These data surprisingly and unexpectedly demonstrate that administration of the oral vitamin E formulation of the present disclosure is therapeutically and commercially viable, unlike various other similar formulations.

[0238] Example 11: Pharmacokinetic study of a vitamin E-containing liraglutide formulation (amorphous) in New Zealand White rabbits This example demonstrates that oral formulations of liraglutide, which contain amorphous liraglutide and are exemplary of oral formulations of the present disclosure, have a therapeutically effective pharmacokinetic profile with advantageous properties compared, for example, to parenteral administration of the same polypeptide.

[0239] This example includes an oral formulation of amorphous liraglutide prepared as follows: Commercially available lyophilized bulk liraglutide (amorphous form) was powdered and passed through a 25 micron sieve to obtain a particle size of less than 25 microns. The lyophilized liraglutide (dosages mentioned in Table 15) was then transferred into size 00 enteric coated capsules containing 100 μL of Vitamin E (Sigma Chemical Company) and 50 μL of wheat germ oil. The capsules were then stored at 4° C. until further use.

[0240] Pharmacokinetic analysis was performed in male New Zealand White rabbits weighing 1.5 kg for 5 days. The experimental design is shown in Table 15. The rabbits were fed with normal chow. Plasma samples taken at different time points were analyzed by Liraglutide (LRT) ELISA kit according to the ABBEXA protocol. The kit is based on competitive enzyme-linked immunosorbent assay technology. Antibody anti-liraglutide was pre-coated on a 96-well plate. Standards, test samples, and biotin-conjugated reagent were added to the wells and incubated. A competitive inhibition reaction occurs between biotin-labeled LRT and unlabeled LRT on the pre-coated antibody. HRP-conjugated reagent was then added and the whole plate was incubated. At each step, unbound conjugates were removed with washing buffer. TMB substrate was used to quantitate the HRP enzyme reaction. After adding TMB substrate, only wells with sufficient LRT produced a blue product, which then turned yellow after adding an acidic stop solution. The intensity of the yellow color is inversely proportional to the amount of LRT bound on the plate. The OD at 450 nm is measured spectrophotometrically using a microplate reader, from which the concentration of LRT can be calculated. [Table 15]

[0241] The Liraglutide ELISA protocol of this example is described below. The following reagents were used after being brought to room temperature: (1) Detection Reagent A, B: Detection Reagent A and B were diluted 1:100 with Assay Diluent A and B, respectively. (2) Washing Buffer (30X): Dilution: To make Washing Buffer (1X), add 20 ml of Washing Buffer (30X) to 580 ml of deionized water. This is the working solution. (3) Liraglutide Standard (1 ml): The standard vial was briefly centrifuged and 1 ml of sample diluent was added to the vial to obtain 1000 pg / ml, which was further serially diluted to obtain 333.33 pg / ml, 111.1 pg / ml, 37.03 pg / ml, and 12.34 pg / ml. (4) TMB Substrate Solution: Once the reagents were thawed, they were appropriately aliquoted and not freeze-thawed more than twice. All reagents were diluted immediately before use. If applicable for low volumes, the vials were quickly spun down to obtain a precipitate at the bottom of the vial before dilution. The ELISA was performed according to the following steps: (1) To each pre-designated well, 50 μl of standard, blank, or sample was added. (2) 50 μl of detection reagent A working solution was immediately added to each well and covered with a plate sealer. The plate was gently swirled to ensure adequate mixing and incubated at 37°C for 1 hour. (3) The fluid from each well was aspirated and washed three times using a multichannel pipette by adding approximately 350 μl of wash buffer. (4) Each wash was performed for 1-2 minutes before aspirating completely. (5) After the final wash, any remaining wash solution was aspirated and the plate was then blotted onto clean absorbent paper. (6) To all wells, 100 μl of detection reagent B working solution was added, gently swirled to ensure adequate mixing, the plate was covered with a new plate sealer, and incubated at 37°C for 30 minutes. (7) The liquid from each well was aspirated and washed three times by adding approximately 350 μl of wash buffer using a multichannel pipette. (8) 90 μl of TMB substrate solution was added to each well and the plate was incubated in the dark at 37°C for 10 to 20 minutes. (9) After incubation, 50 μl of stop solution was added to each well in the same order and at the same time as the TMB substrate solution, and the plate was gently agitated.(10) The optical density (OD value) of each well was immediately read using a microplate reader set at 450 nm. The results are shown in Tables 16 to 27. [Table 16-1] [Table 16-2] [Table 16-3] [Table 16-4] [Table 16-5] [Table 16-6] [Table 17] [Table 18] [Table 19-1] [Table 19-2] [Table 20-1] [Table 20-2] [Table 21] [Table 22] [Table 23-1] [Table 23-2] [Table 24-1] [Table 24-2] [Table 25] [Table 26-1] [Table 26-2] [Table 27-1] [Table 27-2]

[0242] Other embodiments While a number of embodiments have been described, it is apparent that the basic disclosure and examples of the inventors may provide other embodiments that utilize or are encompassed by the compositions and methods described herein, and therefore, it will be understood that the scope is defined not by the specific embodiments shown by way of example, but by what can be understood from this disclosure and the appended claims.

[0243] All documents cited herein are hereby incorporated by reference.

Claims

1. 1. A polypeptide formulation for oral delivery comprising: (i) a crystallized or amorphous polypeptide composition; and (ii) a pharma- ceutical acceptable carrier comprising a vitamin E agent, Optionally, (i) the polypeptide formulation is an encapsulated polypeptide formulation comprising a core and a pharma- ceutically acceptable capsule, wherein the core comprises the crystallized or amorphous polypeptide composition and the pharma- ceutically acceptable carrier, or (ii) the polypeptide formulation is a suspension formulation.

2. 1. A polypeptide formulation for use in a method of delivering a polypeptide to the bloodstream of a subject, the method comprising: (i) a crystallized or amorphous polypeptide composition; and (ii) a pharma- ceutical acceptable carrier comprising a vitamin E agent, the method comprising orally administering the polypeptide formulation to the subject; Optionally, (i) the polypeptide formulation is an encapsulated polypeptide formulation comprising a core and a pharma- ceutically acceptable capsule, wherein the core comprises the crystallized or amorphous polypeptide composition and the pharma- ceutically acceptable carrier, or (ii) the polypeptide formulation is a suspension formulation.

3. 3. The polypeptide formulation of claim 2, wherein the polypeptide is delivered to the bloodstream via the lymphatic system, and optionally, the polypeptide is delivered to the bloodstream via the thoracic duct.

4. 1. A polypeptide formulation for use in a method of delivering a polypeptide to the thoracic duct or lymph of a subject, the method comprising: (i) a crystallized or amorphous polypeptide composition; and (ii) a pharma- ceutical acceptable carrier comprising a vitamin E agent, the method comprising orally administering the polypeptide formulation to the subject; Optionally, (i) the polypeptide formulation is an encapsulated polypeptide formulation comprising a core and a pharma- ceutically acceptable capsule, wherein the core comprises the crystallized or amorphous polypeptide composition and the pharma- ceutically acceptable carrier, or (ii) the polypeptide formulation is a suspension formulation.

5. A method for producing an encapsulated polypeptide formulation for oral delivery, the method comprising encapsulating within a capsule a core comprising (i) an amorphous or crystallized polypeptide composition and (ii) a pharma- ceutically acceptable carrier comprising a vitamin E agent.

6. 1. A method for producing a polypeptide suspension for oral administration, the method comprising: (i) suspending a crystallized or amorphous polypeptide composition in (ii) a pharma- ceutical acceptable carrier comprising a vitamin E agent.

7. The polypeptide, (i) a therapeutic polypeptide, (ii) an antibody drug or a fragment thereof; (iii) a monoclonal antibody or a fragment thereof; (iv) a fusion polypeptide, (v) immunoglobulins, (vi) an enzyme, or (vii) The polypeptide formulation or method of any one of claims 1 to 6, comprising analogs and / or modified forms of any of (i) to (vi), wherein optionally the polypeptide is modified by one or more of the following: pegylation, acetylation, amidation, lipidation, methylation, phosphorylation, glycosylation, glycation, sulfation, mannosylation, nitrosylation, acylation, palmitoylation, prenylation, fatty acids, or combinations thereof.

8. The polypeptide can be an antibody (e.g., an anti-HER2 antibody, e.g., trastuzumab), a GLP-1 receptor agonist, human glucagon-like peptide-1 (GLP-1) or an analog thereof (e.g., a synthetic analog, e.g., liraglutide), a parathyroid hormone (e.g., a recombinant human parathyroid hormone analog, e.g., teriparatide), an insulin (e.g., Humulin®), rituximab, bevacizumab, cetuximab, etanercept, infliximab, agalsidase beta, agalsidase alpha, imiglucerase, aliglucerase alpha, velaglucerase alpha, alglucerase, sebelipase alpha, laronidase, idursulfase, elosulfase alpha, gallus 7. The polypeptide formulation or method of any one of claims 1 to 6, wherein the polypeptide is selected from factor I, factor II, factor III, factor IV, factor V, factor VI, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, von Willebrand factor, etanercept, aflibercept, rilonacept, alefacept, romiplostim, abatacept / belatacept, and / or denileukin-diftitox, or an analogue or derivative thereof, optionally wherein the polypeptide is natural, synthetic or engineered.

9. the polypeptide has a molecular weight of between about 50 Da and about 25 kDa, optionally wherein the molecular weight is between about 50 Da and about 1 kDa, between about 50 Da and about 2 kDa, between about 50 Da and about 3 kDa, between about 50 Da and about 4 kDa, between about 50 Da and about 5 kDa, between about 50 Da and about 10 kDa, between about 50 Da and about 15 kDa, or between about 50 Da and about 20 kDa; Or, the polypeptide formulation or method of any one of claims 1-6, wherein the polypeptide has a molecular weight of between about 25 kDa and about 1,000 kDa, optionally wherein the molecular weight is between about 25 kDa and about 500 kDa, between about 100 kDa and about 500 kDa, between about 120 kDa and about 250 kDa, or between about 150 kDa and about 300 kDa.

10. The polypeptide formulation comprises from about 1 μg to about 2,000 mg of the polypeptide, and optionally the polypeptide formulation comprises from about 1 μg to about 1,000 mg, about 1 μg to about 500 mg, about 1 μg to about 400 mg, about 1 μg to about 300 mg, about 1 μg to about 200 mg, about 1 μg to about 100 mg, about 1 μg to about 50 mg, about 1 μg to about 25 mg, about 1 μg to about 20 mg, about 1 μg to about 15 mg, about 1 μg to about 10 mg, about 1 μg to about 5 mg, about 1 μg to about 1 ...

6. The polypeptide formulation or method of any one of claims 1-5, comprising about μg to about 500 μg, about 1 μg to about 250 μg, about 1 μg to about 200 μg, about 1 μg to about 150 μg, about 1 μg to about 100 μg, about 1 μg to about 50 μg, about 1 mg to about 1,000 mg, about 1 mg to about 500 mg, about 1 mg to about 400 mg, about 1 mg to about 300 mg, about 1 mg to about 200 mg, about 1 mg to about 100 mg, about 1 mg to about 50 mg, or about 1 mg to about 25 mg of the polypeptide.

11. 6. The polypeptide formulation or method of any one of claims 1-5, wherein the polypeptide formulation comprises from about 1 mg to about 2,000 mg of a vitamin E agent, optionally wherein the polypeptide formulation comprises from about 1 mg to about 1,000 mg, from about 1 mg to about 500 mg, from about 1 mg to about 400 mg, from about 1 mg to about 300 mg, from about 1 mg to about 200 mg, from about 1 mg to about 100 mg, from about 1 mg to about 50 mg, or from about 1 mg to about 25 mg of a vitamin E agent.

12. 6. The polypeptide formulation or method of any one of claims 1-5, wherein the polypeptide formulation comprises a molar or weight excess of the vitamin E agent relative to the amount of polypeptide in the polypeptide formulation, optionally wherein the excess is at least a 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, or 1,000 fold excess.

13. 7. The polypeptide formulation or method of any one of claims 1-6, wherein the crystallized polypeptide composition comprises polypeptide crystals having an average particle size of less than 25 microns, e.g., less than 20, 15, 10, 5, 4, 3, 2, 1, or 0.5 microns, e.g., optionally, the polypeptide crystals have an average particle size of between 0.5 microns and 1, 2, 3, 4, 5, 10, 15, 20, or 25 microns, or the polypeptide crystals have an average particle size of between 1 micron and 1, 2, 3, 4, 5, 10, 15, 20, or 25 microns.

14. 7. The polypeptide formulation or method of any one of claims 1-6, wherein the amorphous polypeptide composition comprises crystals of polypeptide having an average particle size of less than 25 microns, e.g., less than 20, 15, 10, 5, 4, 3, 2, 1, or 0.5 microns, and optionally wherein the particles of the polypeptide have an average particle size of between 0.5 microns and 1, 2, 3, 4, 5, 10, 15, 20, or 25 microns, or wherein the particles of the polypeptide have an average particle size of between 1 micron and 1, 2, 3, 4, 5, 10, 15, 20, or 25 microns.

15. The polypeptide formulation or method of any one of claims 1 to 6, wherein the polypeptide composition comprises a lyophilized polypeptide.

16. The polypeptide formulation or method of any one of claims 1 to 6, wherein the polypeptide composition comprises microcrystals of the polypeptide.

17. 7. The polypeptide formulation or method of any one of claims 1 to 6, wherein the polypeptide composition comprises a powder comprising crystallized polypeptide.

18. The polypeptide formulation or method of any one of claims 1 to 5, wherein the core is a viscous solution.

19. 6. The polypeptide formulation or method of any one of claims 1 to 5, wherein the capsule is a hard shell capsule or a soft shell capsule, optionally wherein the capsule is a gelatin capsule or an HPMC capsule.

20. 6. The polypeptide formulation or method of any one of claims 1 to 5, wherein the capsule is formulated for delivery to the intestine, optionally wherein the capsule is formulated for delivery to the stomach and / or intestine.

21. The polypeptide formulation or method of any one of claims 1 to 5, wherein the capsule comprises an enteric coating.

22. the core further comprises one or more excipients or additives selected from the group consisting of an aggregation reducing agent, a sugar or sugar alcohol, a polysaccharide, a stabilizer, a hyaluronidase, a buffering agent, a preservative, a carrier, an antioxidant, a chelating agent, a natural or synthetic polymer, a cryoprotectant, a lyoprotectant, a surfactant, a bulking agent, an acidifying agent, a component that reduces injection site discomfort, an antifoaming agent, a chelating agent, a natural or synthetic polymer, a cryoprotectant, a cryoprotectant, a surfactant, a bulking agent, an acidifying agent, a component that reduces injection site discomfort, an antifoaming agent, an alkalizing agent, a vehicle, an aggregation inhibitor, a solubilizing agent, a tonicity adjusting agent, and a stabilizer, and combinations thereof; 6. The polypeptide formulation or method of any one of claims 1 to 5, optionally wherein the one or more excipients or additives, individually or cumulatively, are present at a concentration of between 0.1 mM and about 1,000 mM, between about 0.1 mM and about 500 mM, between about 0.1 mM and about 200 mM, or between about 0.1 mM and about 100 mM.

23. (i) the aggregation reducing agent(s) is selected from the group consisting of nicotinic acid, caffeine citrate, caffeine nicotinate, caffeine, octyl-β-D-glucopyranoside, and n-dodecyl-β-D-maltoside, optionally in combination with arginine, tryptophan, histidine, proline, cysteine, methionine, β-alanine, potassium glutamate, arginine ethyl ester, lysine, aspartic acid, glutamic acid, glycine, DTPA (diethylenetriaminepentaacetic acid), EGTA (aminopolycarboxylic acid), EDTA (ethylenediaminetetraacetic acid), hydroxypropyl beta (H in combination with one or more of N-(N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid (NBD-X), methyl acetyl phosphate (MAP), citraconic anhydride, pyrophosphate, citrate, and combinations thereof; (ii) the tonicity adjusting agent(s) is selected from the group consisting of arginine, cysteine, histidine, glycine, sodium chloride, potassium chloride, sodium citrate, a sugar (e.g., sucrose, glucose, dextrose, glycerin, or mannitol), and combinations thereof; (iii) the antioxidant(s) are selected from the group consisting of glycine, lysine, EDTA, DTPA, sorbitol, mannitol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, sodium thiosulfate, sulfur dioxide, tocopherol, and combinations thereof; or 23. The polypeptide formulation or method of claim 22, wherein the lyoprotectant(s) is selected from the group consisting of sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, maltose, lactulose, maltulose, glucitol, maltitol, lactitol, isomaltulose, and mannitol; amino acids (e.g., arginine, histidine, proline, or glycine); lyotropic salts (e.g., magnesium sulfate); propylene glycol, glycerol, poly(ethylene glycol), or poly(propylene glycol); gelatin, dextrin, modified starch, carboxymethylcellulose, and combinations thereof.

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