Formulations for oral delivery of polypeptides, antibodies and proteins and their use

Abstract

Nanoparticles comprising a composition comprising a polypeptide having a molecular weight greater than 50,000 g / mol (e.g., IgY antibody), wherein the composition is encapsulated in a material comprising a biocompatible biodegradable polymer, are provided. Also provided are methods of preparing these nanoparticles and the use of the nanoparticles as a therapeutic agent for treating a disease state.

Description

Technical Field

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 341,727, filed May 13, 2022, the content of which is hereby incorporated by reference in its entirety.

[0002] Technical Field The present invention relates to nanoparticles comprising a composition comprising a polypeptide (e.g., an antibody such as an IgY antibody, and a protein) having a molecular weight greater than 50,000 g / mol, wherein the composition is encapsulated in a material comprising a biocompatible and biodegradable polymer. Also provided are methods of preparing these nanoparticles and the use of the nanoparticles as therapeutic agents for treating disease states.

Background Art

[0003] One of the challenges with oral delivery of therapeutic proteins such as IgY antibodies is that most proteins have a shorter lifespan and very little absorption in the gastrointestinal tract. Thus, there is a need for improved formulations of therapeutic proteins such as IgY antibodies for oral delivery.

Summary of the Invention

[0004] In certain embodiments, the present disclosure relates to nanoparticles comprising a composition comprising a polypeptide, an antibody, or a protein, wherein the composition is encapsulated in a material containing a biocompatible biodegradable polymer and a surfactant polymer. In certain embodiments, the biocompatible biodegradable polymer is polymethacrylate. In certain embodiments, the nanoparticles have a diameter of less than 300 nm. In certain embodiments, the nanoparticles comprise at least 5% w / w of the polypeptide. In certain embodiments, the polypeptide is IgY. In certain embodiments, the IgY is obtained from hyperimmunized eggs. In certain embodiments, the composition is a hyperimmunized egg product. In certain embodiments, the composition is a defatted fraction from egg yolk containing at least 95 wt% IgY. In certain embodiments, the composition is purified IgY.

[0005] In certain embodiments, the present disclosure relates to a pharmaceutical composition comprising the nanoparticles described herein and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutically acceptable carrier is suitable for oral administration. The pharmaceutical composition can be in a form selected from the group consisting of powders, tablets, enteric-coated tablets, capsules, enteric-coated capsules, suspensions, solutions, and oral beverages. Substances for forming a controlled release coating can be used to provide an enteric coating for producing enteric-coated tablets or capsules. For example, the substance can be one or more selected from the group consisting of hydroxypropylmethylcellulose phthalate, hydroxymethylethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, carboxymethylethylcellulose, methacrylic acid-methyl methacrylate copolymer (e.g., EUDRAGIT® L 100 and EUDRAGIT® S1 00, Evonik), and methacrylic acid-ethyl acrylate copolymer (e.g., EUDRAGIT® L 100-55 and EUDRAGIT® L 30D55, Evonik).

[0006] In certain embodiments, the present disclosure relates to a method of delivering a polypeptide to a subject, the method comprising administering to the subject, by oral administration, a pharmaceutical composition comprising nanoparticles containing the polypeptides described herein or nanoparticles containing polypeptides. In certain embodiments of the foregoing method, the polypeptide is IgY. In certain embodiments, the IgY is obtained from hyperimmunized eggs. In certain embodiments, the composition in the nanoparticles is a hyperimmunized egg product. In certain embodiments, the composition in the nanoparticles is a defatted fraction from egg yolk containing at least 95% by weight of IgY. In certain embodiments, the composition contained in the nanoparticles comprises purified IgY. In certain embodiments, the biocompatible biodegradable polymer of the nanoparticles comprises polymethacrylate. In certain embodiments, the nanoparticles have a diameter of less than 300 nm. The particles can be used for oral delivery of the polypeptide. The nanoparticles can be included in tablets, capsules, suspensions, emulsions, or beverages for oral administration. In certain embodiments, the nanoparticles comprise at least 5 wt% of the polypeptide based on the total weight of the nanoparticles.

[0007] In certain embodiments, the nanoparticles further comprise a surfactant polymer. The surfactant polymer can be selected from poly(vinyl) alcohol (PVA), polyvinylpyrrolidone (PVP), and combinations thereof.

[0008] Also provided is a method of medical treatment comprising administering to a patient an effective amount of nanoparticles containing a polypeptide.

[0009] Also provided is a method for producing nanoparticles comprising a polypeptide having a molecular weight greater than 50,000 g / mol, the method comprising the steps of mixing a solvent with the polypeptide to form a mixture, mixing the mixture with a biocompatible biodegradable polymer, a surfactant polymer, and an organic solvent, performing emulsification to generate an emulsion, high-pressure homogenizing the emulsion at a pressure of at least 10,000 psi, and evaporating the solvent to leave the nanoparticles, wherein the surfactant polymer is used to stabilize the emulsion.

[0010] Also provided are nanoparticles containing a composition comprising a polypeptide, wherein the composition is encapsulated in a material comprising a biocompatible biodegradable polymer and a surfactant polymer. The biocompatible biodegradable polymer can include polymethacrylate. The biocompatible biodegradable polymer can include a methacrylic acid-methyl methacrylate copolymer (1:1) or a methacrylic acid-methyl methacrylate copolymer (1:2), or a combination thereof. The nanoparticles can have a diameter of less than 300 nm. The nanoparticles can comprise or contain at least 5 wt% polypeptide based on the total weight of the nanoparticles. The polypeptide of the nanoparticles can be IgY. IgY can be obtained from highly immunized eggs. The composition within the nanoparticles can be a highly immunized egg product. The composition within the nanoparticles can be a defatted fraction from egg yolk containing at least 95 wt% IgY. The composition within the nanoparticles can be or include purified IgY. In the nanoparticles provided herein, the surfactant polymer can be selected from poly(vinyl) alcohol (PVA), polyvinylpyrrolidone (PVP), and combinations thereof.

[0011] Also provided is a method for delivering a polypeptide, antibody, or protein, the method comprising administering to a subject, by oral administration, a pharmaceutical composition comprising the nanoparticles described herein. The polypeptide contained in the nanoparticles can be IgY. IgY can be obtained from hyperimmunized eggs. The nanoparticles can contain a composition comprising a hyperimmunized egg product. The nanoparticles can contain a composition comprising a defatted fraction from egg yolk containing at least 95% by weight of IgY. The nanoparticles can contain a composition comprising purified IgY. In this method, the delivered nanoparticles can comprise a biocompatible and biodegradable polymer. The biocompatible and biodegradable polymer can comprise polymethacrylate. The biocompatible and biodegradable polymer can comprise a methacrylic acid-methyl methacrylate copolymer (1:1) or a methacrylic acid-methyl methacrylate copolymer (1:2), or a combination thereof. The nanoparticles can have a diameter of less than 300 nm. The nanoparticles can contain at least 5% w / w of polypeptide based on the total weight of the nanoparticles.

[0012] Also provided is a method for preparing the nanoparticles described in this specification. The method includes the steps of: (a) preparing a first mixture comprising a composition containing a polypeptide, water, and a surfactant polymer; (b) subjecting the first mixture to sonication; (c) adding a biocompatible biodegradable polymer to ethanol and subjecting it to sonication to produce a second mixture; (d) mixing the first mixture and the second mixture with an organic solvent to form a third mixture; (e) homogenizing the third mixture using a probe homogenizer at a speed of about 5,000 - 15,000 rpm to produce a crude emulsion; (f) subjecting the crude emulsion to high-pressure homogenization at a pressure of about 10,000 psi - 30,000 psi using a high-pressure homogenizer to form a fourth mixture; and (g) evaporating ethanol and the organic solvent from the fourth mixture to produce a suspension containing nanoparticles. The method may also include, as a step, centrifuging the suspension to obtain the nanoparticles. In this method, the surfactant polymer can be selected from poly(vinyl) alcohol (PVA), polyvinylpyrrolidone (PVP), and combinations thereof. The organic solvent may include absolute ethanol, dichloromethane (DCM), acetonitrile, acetone, ethyl acetate, chloroform, and combinations thereof. In some methods, the organic solvent includes dichloromethane. The first mixture may contain about 2 wt% - 5 wt% of the polypeptide, and the polypeptide may be IgY. The second mixture may contain 10 mg / mL - 20 mg / mL of the biocompatible biodegradable polymer. In some embodiments, the surfactant polymer includes 0.5 - 2.5 wt% of poly(vinyl) alcohol (PVA), or 0.5 - 2.5 wt% of polyvinylpyrrolidone (PVP), or a combination thereof. The first mixture may contain 0.75 wt% - 2 wt% of PVA.

Brief Description of the Drawings

[0013]

Figure 1

Best Mode for Carrying Out the Invention

[0014] In certain embodiments, the present disclosure relates to nanoparticles containing a composition comprising a polypeptide, wherein the composition is encapsulated in a biocompatible biodegradable polymer and a surfactant polymer that exhibits at least weak surfactant activity. Examples of surfactant polymers include polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), and combinations thereof. Applicants have shown that polypeptides having a molecular weight greater than 50,000 g / mol, or greater than 100,000 g / mol, such as IgY, encapsulated in nanoparticles comprising a biocompatible biodegradable polymer (e.g., EUDRAGIT® L 100 or EUDRAGIT® S 100 (Evonik Industries AG, Essen, Germany)) or combinations thereof and a surfactant polymer, remain intact through the encapsulation process and have identified an encapsulation method that results in reduced nanoparticle size and high drug loading of the nanoparticles. In certain embodiments, the encapsulated composition is a highly immunized egg product and the polypeptide is an IgY antibody. In some embodiments, the nanoparticles contain a composition comprising a purified IgY antibody.

[0015] Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, patent applications, published applications and publications, websites, and other published materials referred to throughout this specification are incorporated by reference in their entirety unless otherwise described. When there are multiple definitions for a term in this specification, the definitions in this section shall prevail. When a URL or other such identifier or address is referred to, it is understood that such identifiers may change and that specific information on the Internet may appear or disappear, but equivalent information can be found by searching the Internet. References thereto serve as evidence that such information is available and publicly disseminated.

[0016] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0017] As used herein, all ranges include upper and lower limits. As used herein, a description of a numerical range for a variable is intended to convey that the variable can be equal to any value within the range as well as any sub-range encompassed by a broader range. Thus, the variable can be equal to any integer value or value within the numerical range, including the endpoints of the range. By way of example, a variable described as having a value between 0 and 10 can be 0, 4, 2 - 6, 2.75, 3.3 - 4.4, etc.

[0018] As used herein, "about" is an approximate term and is intended to include minor variations from the precisely stated amount as would be understood by one of ordinary skill in the art. Such variations include, for example, standard deviations associated with the amounts of components or ingredients of a mixture or composite material, or other properties and characteristics measured by techniques generally used. All values characterized by the modifier "about" are also intended to include exactly that numerical value in combination therewith. Thus, "about 5 percent" means "about 5 percent" and also "5 percent".

[0019] As used herein, the terms "comprises" and "comprising" are inclusive and open-ended and not exclusive. When used in this specification and the claims, the terms "comprises" and "comprising" and their variations mean that the specified feature, step or component is included, but do not exclude other features, steps or components.

[0020] Unless otherwise expressly stated to the contrary, all compositions described herein are intended to include compositions consisting of, consisting essentially of, and comprising the various components specified herein.

[0021] In this specification and the claims, the singular form includes the plural unless the context clearly dictates otherwise. As used herein, unless otherwise indicated specifically, the word "or" is used in the "inclusive" sense of "and / or" and not in the "exclusive" sense of "either / or".

[0022] As used herein, the term "exemplary" means "serving as an example or illustration" and should not be construed as being preferred or advantageous over other configurations disclosed herein.

[0023] Unless otherwise indicated, each individual feature or embodiment of this specification can be combined with any other individual feature or embodiment described herein without limitation. Such combinations are specifically contemplated to be within the scope of the present invention, whether or not they are explicitly described as combinations herein.

[0024] As used herein, the term "subject" includes members of the animal kingdom including, but not limited to, humans.

[0025] As used herein, "weight percent" or "wt%" refers to the concentration of a substance obtained by dividing the weight of the substance by the total weight of the composition and multiplying by 100.

[0026] The term "hyperimmunization" means repeated exposure to one or more antigens such that the immune response is elevated and maintained beyond the natural non-exposed state.

[0027] "Hyperimmune state" refers to an elevated immune response in an egg-laying animal that has been hyperimmunized.

[0028] As used herein, the term "egg" refers to whole eggs (edible, hyperimmunized or otherwise). The term "egg product" as used herein refers to whole eggs or any product (product) or fraction obtained from whole eggs. In certain embodiments, the egg product is egg yolk, such as egg yolk powder. In another embodiment, the egg product is egg white, such as egg white powder. In another embodiment, the egg product is obtained from whole eggs, such as whole egg powder (e.g., spray-dried whole egg powder).

[0029] The term "control egg" refers to an egg obtained from an egg-laying animal that is not maintained in a hyperimmune state, i.e., an animal that has not been hyperimmunized. The term "control egg product" refers to a control egg or an egg product obtained from a control egg.

[0030] The term "hyperimmunized egg" refers to whole eggs obtained from egg-laying animals maintained in a hyperimmunized state, i.e., egg-laying animals that have been hyperimmunized. The term "hyperimmunized egg product" refers to hyperimmunized eggs or any product obtained from hyperimmunized eggs.

[0031] In certain embodiments, the hyperimmunized egg product is a concentrate. As used herein, the term "concentrate" refers to a hyperimmunized egg product that has been at least partially purified such that the concentration of antibodies in the concentrate is greater than the concentration of antibodies in the hyperimmunized eggs.

[0032] The term "egg powder" refers to dried whole eggs. In some embodiments, the egg powder is spray-dried.

[0033] The term "egg-laying animal" means any oviparous animal and includes any animal that lays eggs, such as birds, fish, and reptiles.

[0034] The term "bird" refers to an animal that is a member of the class Aves. Birds include, but are not limited to, chickens, turkeys, geese, ducks, pheasants, quails, pigeons, and ostriches.

[0035] The term "administer" means any method of providing a substance to a subject, including oral, intranasal, parenteral (intravenous, intramuscular, or subcutaneous), rectal, topical, or intraocular.

[0036] The term "antigen" refers to a substance that can induce a humoral antibody and / or cell-mediated immune response, rather than immunological tolerance. This term also means the ability not only to stimulate an immune response but also to react with the products thereof, such as antibodies.

[0037] As used herein, "antibody" refers to a protein that includes at least one complementarity determining region that binds to a specific target antigen. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. In certain embodiments, the antibody is a polyclonal antibody. In certain embodiments, the antibody is an IgY antibody.

[0038] As used herein, "IgY" refers to one or more IgY antibodies.

[0039] The term "polyclonal antibody" as used herein refers to a population of antibody molecules that can immunoreact with different epitopes on a specific antigen.

[0040] As used herein, "nanoparticle" refers to a particle or structure in the nanometer (nm) range, typically having a diameter of about 1 to about 1000 nm.

[0041] As used herein, the term "loading percentage" refers to 100 multiplied by the ratio of the weight of a polypeptide, such as an IgY antibody, to the weight of the nanoparticle.

[0042] As used herein, "dalton" is a unit of molecular weight abbreviated as Da, and 1 Da is equal to 1 g / mol.

[0043] As used herein, "polypeptide" refers to a polymer that includes a plurality of linked amino acid residues and has a molecular weight of at least 50,000 Da (g / mol) or more.

[0044] As used herein, "protein" refers to a polymer that includes a plurality of linked amino acid residues and has a molecular weight of at least 50,000 Da (g / mol) or more.

[0045] The IgY antibody molecule has a structure similar to that of the IgG antibody and has two heavy chains (H), each having a molecular weight of about 67 - 70 kDa, and two light chains (L) having a molecular weight of about 25 kDa.

[0046] As used herein, "probe homogenizer" refers to a rotor / stator type homogenizer that includes a rotating blade member (rotor) and a stationary collar (stator) containing perforations.

[0047] As used herein, "high-pressure homogenizer" refers to a homogenizing device that includes a homogenization valve typically containing a narrow channel through which a material is forced to flow, and a high-pressure pump that pushes the material through the valve. The high-pressure homogenizer typically operates at a pressure of 5,000 psi to 45,000 psi.

[0048] Highly immunized egg products Oviparous animals produce antibodies specific to certain immunogens in their blood and eggs. For example, various genera of the class Aves, such as chickens (Gallus domesticus), turkeys, and ducks, produce antibodies against antigens associated with avian diseases. LeBacq-Verheyden et al. (Immunology 27:683 (1974)) and Leslie, G.A. et al. (J. Med. 130:1337 (1969)) quantitatively analyzed chicken immunoglobulins. Polson, A. et al. (Immunological Communications 9:495-514 (1980)) immunized hens against several proteins and natural mixtures of proteins and detected IgY antibodies in egg yolk. Fertel, R. et al. (Biochemical and Biophysical Research Communications 102:1028-1033 (1981)) immunized hens against prostaglandins and detected antibodies in egg yolk. Jensenius et al. (Journal of Immunological Methods 46:63-68 (1981)) provided a method for isolating egg yolk IgG for use in immunodiagnosis. Polson et al. (Immunological Communications 9:475-493 (1980)) described antibodies isolated from the egg yolk of hens immunized with various plant viruses.

[0049] U.S. Patent No. 4,748,018 (Stolle et al., 1988) discloses a method for passive immunization of mammals, which method comprises parenterally administering a purified antibody obtained from the eggs of birds immunized against the corresponding antigen, and the mammal acquires immunity to the eggs. U.S. Patent No. 5,772,999 (Greenblatt et al., 1998) discloses a method for preventing, counteracting, or reducing chronic gastrointestinal disorders or non-steroidal anti-inflammatory drug (NSAID)-induced gastrointestinal damage in a subject by administering highly immunized eggs and / or milk or fractions thereof to the subject.

[0050] Immunized eggs are eggs derived from birds immunized, for example, with a specific antigen or a mixture of antigens. Hyperimmunized eggs are eggs derived from birds that have been brought into a specific immunized state, for example, by periodic booster administrations of an antigen. Hyperimmunized eggs, regardless of the type of antigen administered by the bird manufacturer, have been found to have various beneficial factors as described above, including the treatment of chronic gastrointestinal disorders, NSAID-induced gastrointestinal injury (see U.S. Patent No. 5,772,999), and anti-inflammatory effects due to the presence of anti-inflammatory compositions (see U.S. Patent Application Publication No. US2004 / 0156857 (Adalsteinsson et al., 2004)).

[0051] One advantage of hyperimmunized egg products is that they will have higher and more consistent levels of antibodies (e.g., IgY antibodies) against one or more of the antigens described herein compared to control egg products or egg products from chickens immunized with an antigen using standard immunization techniques. Typically, standard immunization consists of a primary immunization followed by one or two booster immunizations at 30-day intervals. In some embodiments, hyperimmunization includes at least 4, 5, 6, 7, 8, 9, or 10 immunizations with the antigens described herein. In some embodiments, hyperimmunization includes immunizing the laying animal with the antigens described herein at intervals of less than 30 days, less than 25 days, less than 20 days, less than 15 days, less than 10 days, or less than 5 days. In some embodiments, hyperimmunization includes immunizing the laying animal with the antigens described herein at intervals of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, or 3 months. Any of these values can be used to define the range of intervals at which the laying animal is immunized. For example, in some embodiments, the laying animal is hyperimmunized at intervals ranging from once every 2 weeks to once every 3 months, once every 1 week to once every 3 months, or once every 2 weeks to once every 1 month.

[0052] Highly immunized egg products may be produced by any egg-laying animal. The animal is preferably a member of the avian class or, in other words, a bird. Among the avian class, domesticated poultry is preferred, but other members of this class, such as turkeys, ducks, and geese, are suitable sources of highly immunized egg products. In certain embodiments, the egg-laying animal is a chicken.

[0053] This special highly immunized state is preferably achieved by initially immunizing and subsequently administering regular boosters with a sufficiently high dose of a specific antigen or mixture of antigens. The booster dosage can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the dosage required to effect primary immunization of the egg-laying animal. Any of these percentages can be used to define the range of booster immunization dosages. For example, in some embodiments, the booster dosage is 20% - 80%, 30% - 70%, or 50% - 100% of the dosage required to effect primary immunization of the egg-laying animal. In certain embodiments, the booster immunization dosage is 50% of the primary immunization dosage.

[0054] With knowledge of the requirements for generating and maintaining a highly immunized state, it is within the skill of the art to vary the amount of antigen administered to maintain an egg-laying animal in a highly immunized state, depending on the genus and strain of the egg-laying animal used.

[0055] The highly immunized state can be brought about by a single antigen or a combination of antigens. Hyperimmunization can be achieved by multiple exposures to multiple antigens or multiple exposures to a single antigen.

[0056] Hyperimmunization procedure The following list of steps is an example of a preferred procedure that can be used to hyperimmunize an egg-laying animal and administer the resulting highly immunized eggs or egg products to birds: 1. Select one or more antigens. 2. Induce an immune response in egg-laying animals by a single immunization. 3. Administer a booster vaccine of one or more antigens in an appropriate dosage to induce and maintain a highly immune state.

[0057] Step 1: The key point in this step is that the antigen must be able to induce an immune state and a highly immune state in egg-laying animals.

[0058] Step 2: The vaccine can be administered by any method that induces an immune response. Immunization is preferably achieved by administering the vaccine via intramuscular or subcutaneous injection. The preferred muscle for injection in birds is the pectoral muscle. The dosage is preferably an immunogenic vaccine of 0.05 to 5 milligrams. Other administration methods that can be used include intravenous injection, intraperitoneal injection, intradermal, rectal suppository, aerosol, or oral administration.

[0059] It is possible to determine whether the vaccine has induced an immune response in egg-laying animals by several methods known to those skilled in immunology. Examples of these include the enzyme-linked immunosorbent assay (ELISA), tests for the presence of antibodies against the stimulating antigen, and tests designed to evaluate the ability of immune cells from the host to respond to the antigen. The minimum dosage of antigen required to induce an immune response depends on the vaccination procedure used, including the type of adjuvant used, the formulation of the antigen, and the type of egg-laying animal used as the host.

[0060] Step 3: The highly immune state is preferably induced and maintained in the target animal by repeated booster administrations of appropriate dosages at fixed time intervals. The time intervals are preferably between 1 week and 3 months over a period of 6 to 12 months. However, it is essential that the booster administration does not cause immune tolerance. Such a process is well known in the art. Methods for preparing highly immunized egg products are described, for example, in U.S. Patent No. 6,803,035 (Greenblatt et al., 2004), which is hereby incorporated by reference in its entirety.

[0061] In certain embodiments, the antigens described herein are formulated into vaccines containing adjuvants. The adjuvants can be selected from Freund's complete adjuvant, Freund's incomplete adjuvant, saponin, biodegradable polymers, aluminum hydroxide, mineral oil, surfactants, and combinations thereof. Exemplary saponins include QS-21 and Quil A. Exemplary biodegradable polymers include chitosan, zymosan, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), such as Pluronic® L121 block copolymer, poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), polycaprolactone, and combinations thereof. Exemplary surfactants include polysorbate 80 and sorbitan trioleate. In some embodiments, the antigens described herein are formulated with an adjuvant selected from the group consisting of Freund's complete adjuvant, Freund's incomplete adjuvant, and QS-21 saponin. In the initial vaccination, laying animals receive two 0.5 mL doses of each antigen. Two weeks later, one 0.5 mL dose of each antigen is administered to the laying animals as a booster vaccination. Additional booster vaccinations are performed 4 weeks after the initial vaccination. The vaccine can be administered intramuscularly. The vaccine can be administered to the mammary tissue.

[0062] Other procedures for maintaining hyperimmunization or combinations of procedures are possible, for example, intramuscular injection for primary immunization and intravenous injection for booster injection. Further procedures include co - administration of microencapsulated antigen and liquid antigen, or intramuscular injection for primary immunization and booster dosing by oral or parenteral administration by means of microencapsulation. Some combinations of primary and hyperimmunization are known to those skilled in the art.

[0063] Hyperimmunized eggs or hyperimmunized egg products may contain increased levels of antibodies (e.g., IgY antibodies) specific to the particular antigens disclosed herein, as compared to control eggs or control egg products obtained from egg - laying animals not hyperimmunized with the particular antigen. In certain embodiments, the antibody is an IgY antibody.

[0064] In some embodiments, the hyperimmunized eggs or egg products contain at least 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400% or 500% more, by weight, of antibodies (e.g., IgY antibodies) specific to the particular antigens disclosed herein, as compared to control eggs or control egg products obtained from egg - laying animals not hyperimmunized with the particular antigen.

[0065] Hyperimmunized eggs or hyperimmunized egg products may contain increased levels of antibodies against two or more of the antigens disclosed herein, as compared to control eggs or control egg products obtained from egg - laying animals not hyperimmunized.

[0066] The comparison of antibody titers in highly immunized egg products and control egg products can be determined by methods known in the art. For example, in one embodiment, eggs are collected and antibody titers are monitored at regular intervals by ELISA. To determine the antibody titer, total IgY is extracted from the eggs using the Pierce™ Chicken IgY Purification Kit (Thermo Fisher Scientific, Waltham, MA). Briefly, 2 mL of eggs are mixed with 5 volumes of defatted reagent and the IgY is purified according to the manufacturer's instructions. The spray-dried egg powder sample is reconstituted at 1 mg / mL in sterile PBS and filtered through a 0.22 μm membrane filter. The specific antibody titer in the isolated IgY or egg powder sample is measured by ELISA. Flat-bottom 96-well microtiter plates (Corning® Costar®, Corning, NY) are coated with 10 μg / mL (100 μL / well) of purified recombinant protein (e.g., antigen B, C, Co1, or Co2) and incubated overnight at 4°C. The plates are washed twice with PBS containing 0.05% Tween 20 (Sigma-Aldrich, St. Louis, MO) and blocked with 100 μL / well of PBS containing 1% bovine serum albumin (BSA) and incubated for 1 hour at room temperature. Serial dilutions of the IgY sample from the egg powder sample (in PBS containing 0.1% BSA) are added to the plates (100 μL / well) in triplicate wells and incubated for 2 hours at room temperature with constant shaking. The plates are then washed with PBS-T, treated with peroxidase-conjugated rabbit anti-chicken IgY (IgG) antibody (1:500, Sigma) and incubated for 30 minutes, followed by development with 0.01% tetramethylbenzidine substrate (Sigma) in 0.05 M phosphate-citrate buffer, pH 5.0 for 10 minutes. Bound antibody is detected by measuring the optical density (OD 450 ) at 450 nm using a microplate reader (Bio-Rad, Hercules, CA).

[0067] The antibody titer can be represented by the maximum dilution multiple of the egg product that still contains detectable antibodies measured by optical density as described above. For example, an antibody titer of 1000 would indicate that a 1000-fold dilution of the egg product contains detectable antibodies, while higher dilutions would not. In some embodiments, the antibody titer in a highly immunized egg product is at least 100,000, at least 250,000, at least 500,000, or at least 1 million, 2 million, 3 million, 4 million, 5 million, 6 million, 7 million, 8 million, 9 million, 10 million, 11 million, 12 million, 13 million, 14 million, 15 million, 16 million, 17 million, 18 million, 19 million, or 20 million.

[0068] In some embodiments, the highly immunized egg or egg product contains at least 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, or 0.1% by weight of IgY antibodies against a specific antigen. Typically, a whole chicken egg weighs about 60 grams without the shell, the egg yolk weighs about 20 grams, and the egg white weighs about 40 grams. In some embodiments, 3 grams of egg yolk contains about 25 milligrams of total IgY, so a whole egg contains about 150 - 200 mg of total IgY. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, or 30% of the total IgY in the highly immunized egg or egg product is specific to one of the antigens used for hyperimmunization.

[0069] Highly immunized eggs or egg products can contain increased levels of two or more antibodies (e.g., IgY antibodies) compared to control eggs or egg products obtained from non-highly immunized laying animals, and each of these antibodies is specific for a different antigen disclosed herein. The increased level of each antibody (e.g., IgY antibody) in the highly immunized eggs or egg products can be at least 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400%, 500% or more by weight compared to the control eggs or egg products.

[0070] Compositions and Administration When laying animals are sufficiently highly immunized, eggs from these animals can be collected and processed to produce a highly immunized egg product in an administrable form. The highly immunized egg product can be prepared by dehydration, spray drying, or freeze drying of whole eggs, egg yolks, or purified IgY fractions. The dried highly immunized egg product can be mixed with an agent, such as silicon or a silicon derivative that improves flow properties. The dried highly immunized egg product may contain a desiccant or may be stored in a container containing a desiccant. The highly immunized egg product may be stored at ambient temperature or may be refrigerated, for example, at 4°C.

[0071] In some embodiments, the highly immunized egg products can be encapsulated. In some embodiments, the nanoparticles provided herein can be encapsulated. Methods of encapsulating antibodies and other proteins are known in the art and are described, for example, in U.S. Patent No. 7,105,158 (D’Souza et al., 2006). Materials that are biodegradable and non-antigenic can be used as encapsulating materials. Examples of encapsulating materials include, but are not limited to, albumin, PLGA, globulin, natural and synthetic polymers, and thermoplastic polymers. Any biocompatible and biodegradable polymer can be used for encapsulation. Some available cross-linking agents, such as glutaraldehyde, can be used to cross-link the encapsulating materials. Further, the nanospheres provided herein may be included with microspheres, and the microspheres are provided as two or more populations, each population of microspheres having a different cross-linking level, thereby providing long-term continuous release of the nanospheres containing polypeptides such as IgY.

[0072] Micro- and Nanoparticle Formulations In certain aspects, the disclosure relates to nanoparticles comprising a composition comprising a polypeptide, wherein the composition is encapsulated in a biocompatible biodegradable polymer and a surfactant polymer such as PVA, polyvinylpyrrolidone, or a combination thereof. The nanoparticles are colloidal particles with a size range in nanometers. Due to their smaller size, the surface area is larger, which tends to result in higher loading efficiency and further enhance the bioavailability of the polypeptide contained within the nanoparticles.

[0073] In some embodiments, the polypeptide is a therapeutic polypeptide, i.e., a polypeptide that is administered to a subject for the treatment of a disorder. In some embodiments, the polypeptide is an antibody, such as a polyclonal antibody or a monoclonal antibody. In some embodiments, the antibody is IgY. In some embodiments, the IgY is obtained from hyperimmunized eggs. In some embodiments, the composition encapsulated in the nanoparticles is a hyperimmunized egg product containing IgY. In some embodiments, the composition contained within the nanoparticles is a defatted fraction from egg yolk containing at least 95 wt%, 96 wt%, 97 wt%, 98 wt%, or 99 wt% IgY based on the total weight of the defatted egg yolk fraction. In some embodiments, the composition contained within the nanoparticles contains purified IgY.

[0074] The nanoparticles can be prepared using one or more polymers. Suitable polymers include, but are not limited to, polyacrylate, polymethacrylate, polycarbonate, polypropylene, polyalkylene, polyalkylene glycol, polyalkylene oxide, polyalkylene terephthalate, polyvinyl ether, polyvinyl halide, polysiloxane, polyurethane and their copolymers, hydroxyalkyl cellulose, cellulose ether, nitrocellulose, methyl cellulose, ethyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose triacetate, sodium salt of cellulose sulfate, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, poly(ethylene terephthalate), poly(vinyl acetate), and polyvinyl chloride, polystyrene, and mixtures, copolymers, and blends thereof. In certain embodiments, the polymer comprises polymethacrylate.

[0075] In some embodiments, the polymer used to form the nanoparticles is EUDRAGIT® L 100 (methacrylic acid copolymer L (methacrylic acid-methyl methacrylate copolymer (1:1), or methacrylic acid copolymer, type A, available from Evonik Corporation, Allentown, PA). The ratio of free carboxyl groups to ester groups is about 1:1 for EUDRAGIT® L 100.

[0076] In some embodiments, the polymer used to form the nanoparticles or microparticles is EUDRAGIT® S 100 (methacrylic acid copolymer S (methacrylic acid-methyl methacrylate copolymer (1:2), or methacrylic acid copolymer, type B, available from Evonik Corporation, Allentown, PA). The ratio of free carboxyl groups to ester groups is about 1:2 for EUDRAGIT® S 100.

[0077] In some embodiments, a combination of EUDRAGIT® L 100 and EUDRAGIT® S 100 can be used to form nanoparticles or microparticles. The ratio of EUDRAGIT® L 100 to EUDRAGIT® S 100 can be in the range of 99:1 to 1:99. In some embodiments, the ratio of EUDRAGIT® L 100 to EUDRAGIT® S 100 can be 80:20 to 20:80, or 75:25 to 25:75, or 60:40 to 40:60. In some embodiments, EUDRAGIT® L 100 is used more than EUDRAGIT® S 100 (there is EUDRAGIT® L 100 with a wt% greater than EUDRAGIT® S 100). In some embodiments, the ratio of EUDRAGIT® L 100 to EUDRAGIT® S 100 is in the range of 2:1 to 10:1. In some embodiments, EUDRAGIT® S 100 is used more than EUDRAGIT® L 100 (there is EUDRAGIT® S 100 with a wt% greater than EUDRAGIT® L 100). In some embodiments, the ratio of EUDRAGIT® S 100 to EUDRAGIT® L 100 is in the range of 2:1 to 10:1. In some embodiments, equal amounts of EUDRAGIT® L 100 and EUDRAGIT® S 100 are used.

[0078] The pH-responsive release profile for the nanoparticles can be achieved by an appropriate selection of one or a combination of the polymers as biocompatible and biodegradable polymers used to form the nanoparticles.

[0079] When forming the nanoparticles or microparticles, a surface-active polymer can also be included. Exemplary surface-active polymers include poly(vinyl) alcohol PVA, polyvinylpyrrolidone (PVP), and combinations thereof. In some embodiments, the surface-active polymer includes PVA.

[0080] By encapsulating IgY in nanoparticles, IgY becomes more stable and can remain in its native structure without being degraded. By using nanoparticles, IgY trapped inside the nanoparticles is safe not only in gastric juice but also in intestinal juice and is protected from enzymatic degradation. Since it is trapped inside the nanoparticles, the molecular weight and large size of IgY are no longer a problem because the overall size of the nanoparticles is in the nano range. Therefore, it can be more easily presented to and pass through the cell or interstitial region and reach the target site.

[0081] The nanoparticles can have any shape. Typically, the nanoparticles are spherical. Other suitable shapes include, but are not limited to, flakes, triangles, ellipses, rods, polygons, needles, tubes, cubes, and rectangular prism structures. In certain embodiments, the nanoparticles have a diameter of less than 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 25, or 10 nm. Any of these values can be used to define the range of the diameter of the nanoparticles. For example, the diameter of the nanoparticles can be about 10 to about 1000 nm, about 100 to about 1000 nm, or about 50 to about 500 nm, or about 10 to about 100 nm, about 200 to about 300 nm, about 250 to about 300 nm, or about 100 to about 400 nm. In certain embodiments, the nanoparticles have a diameter of less than 300 nm.

[0082] The concentration of the polypeptide (e.g., IgY) encapsulated in the nanoparticles can be presented as the loading percentage, i.e., 100 multiplied by the ratio of the weight of the polypeptide (e.g., IgY) to the weight of the nanoparticles. In some embodiments, the nanoparticles contain about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 wt% of the polypeptide (e.g., IgY) relative to the total weight of the nanoparticles. In some embodiments, the nanoparticles contain less than about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 wt% of the polypeptide (e.g., IgY) relative to the total weight of the nanoparticles. In some embodiments, the nanoparticles contain at least about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 wt% of the polypeptide (e.g., IgY) relative to the total weight of the nanoparticles. Any of these values can be used to define the range of the concentration of the polypeptide (e.g., IgY) in the nanoparticles. For example, the nanoparticles can contain the polypeptide (e.g., IgY) at a concentration in the range of about 1 wt% to about 10 wt%, or about 1 wt% to about 5 wt%. In some embodiments, the concentration of the polypeptide (e.g., IgY) in the nanoparticles is at least about 5 wt%.

[0083] In some embodiments, the nanoparticles comprising a polypeptide (e.g., IgY) are anionic and, for example, have a negative zeta potential. In some embodiments, the zeta potential of the nanoparticles is less than 0, -1, -5, -10, -15, -20, -25, -30, -31, -32, -33, -34, -35, -36, -37, -38, -39, -40, -45 or -50 mV. Any of these values can be used to define the range of the zeta potential of the nanoparticles. For example, in some embodiments, the zeta potential of the nanoparticles is from about 0 to about -40 mV, from about 0 to about -50 mV, from about -10 to about -50 mV, from about -20 to about -50 mV, or from about -30 to about -40 mV. In some embodiments, the zeta potential of the nanoparticles is about -38.9 mV. In some embodiments, the zeta potential of the nanoparticles is about -31.2 mV.

[0084] For example, there are several processes by which nanoparticles can be prepared, including multilayer microencapsulation, hot melt encapsulation, phase separation encapsulation, spontaneous emulsification, solvent evaporation microencapsulation, solvent removal microencapsulation, and coacervation. These methods are known in the art. A detailed description of these methods is discussed in Mathiowitz et al., “Microencapsulation”, in Encyclopedia of Controlled Drug Delivery, vol. 2, pp. 495-546, 1999, John Wiley & Sons, Inc.. New York, N.Y., which is hereby incorporated by reference in its entirety.

[0085] A method for preparing nanoparticles comprising a polypeptide having a molecular weight greater than 50,000 g / mol is provided. For example, in some embodiments, the molecular weight of the polypeptide (e.g., IgY) encapsulated in the nanoparticles is at least 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000 or 200,000 g / mol. In some embodiments, the molecular weight of the polypeptide (e.g., IgY) encapsulated in the nanoparticles is 50,000 - 100,000 g / mol, 50,000 - 180,000 g / mol, or 50,000 - 200,000 g / mol. In some embodiments, the molecular weight of the polypeptide (e.g., IgY) encapsulated in the nanoparticles is about 180,000 g / mol.

[0086] A combination of homogenization processes is used to prepare the nanoparticles. A probe homogenizer can be used to create the initial emulsion, which can then be processed with a high-pressure homogenizer.

[0087] The probe homogenizer is a rotor-stator homogenizer that includes a rotating blade member (rotor) and a stationary collar containing perforations (stator). The rotating blade pushes the material through the perforations, and as the material passes through the perforations, the shear force between the perforations in the stator and the rotating blade reduces the particle size. The movement of the blade draws the material into the rotor / stator assembly, which also causes cavitation, which can also help reduce the particle size. The size of the perforations in the stator, the rotational speed of the rotating blade member, the viscosity of the material, and the length of the processing time can affect the resulting particle size. Probe homogenizers with various dimensions and configurations of the rotor and stator are known in the art, commercially available, and can be used in the methods provided herein.

[0088] Next, the emulsion produced using the probe homogenizer can be processed with a high-pressure homogenizer (HPH). The high-pressure homogenizer converts the flow pressure into kinetic energy. A typical high-pressure homogenizer includes a homogenization valve through which the material is forced to flow and a high-pressure pump that pushes the material through the valve. The high-pressure homogenization process exposes the material being processed to multiple forces such as shear, cavitation, turbulent flow, impact force, and high pressure, which can assist in the formation of nanoparticles and result in nanoparticles having optimal characteristics such as minimum particle size, good particle size distribution, and maximum polypeptide loading. High-pressure homogenizers of various dimensions and configurations are known in the art, commercially available, and can be used in the methods provided herein.

[0089] In an exemplary embodiment, the aqueous phase can be prepared to contain a composition comprising a polypeptide (e.g., IgY) and a surfactant polymer. The surfactant polymer can be any one of poly(vinyl) alcohol (PVA), polyvinylpyrrolidone (PVP), or a combination thereof. Next, the organic phase is prepared. The organic phase can include absolute ethanol, dichloromethane (DCM), acetonitrile, acetone, ethyl acetate, chloroform, and combinations thereof. In some methods, the organic phase includes DCM. A solution or suspension containing a biocompatible biodegradable polymer is prepared. To aid mixing, the aqueous phase, or the solution or suspension of the biocompatible biodegradable polymer, or both, can be subjected to sonication. Next, the aqueous phase, the organic phase, and the solution or suspension of the biocompatible biodegradable polymer are mixed together and can be subjected to emulsification using a probe homogenizer (rotor / stator homogenizer). The configuration and duration of mixing can depend on the volume of the material being processed. In some methods, the mixture is subjected to homogenization using a probe homogenizer at a rotor rotation speed of 5,000 to 15,000 rpm. The processing time can be 1 to 10 minutes. A cooling element can be included during probe homogenization. For example, the container containing the material being processed can be placed in an ice bath or a cooling system can be used to remove heat from the material being processed during probe homogenization. Treatment with a probe homogenizer results in a coarse emulsion.

[0090] The coarse emulsion is then subjected to a further homogenization process using a high-pressure homogenizer. The pressure used can be from about 5,000 psi to 45,000 psi. In some embodiments, the method includes treating in a high-pressure homogenizer at a pressure of about 15,000 psi.

[0091] Next, the emulsion obtained from the high-pressure homogenizer can be subjected to solvent removal. In some embodiments, a rotary evaporation apparatus can be used to remove the solvent. Any rotary evaporation apparatus known in the art can be used to remove the solvent. In some embodiments, a rotary vacuum evaporation apparatus can be used. The evaporation time of the solvent can depend on the amount of solvent present and the chemical characteristics of the solvent. In some embodiments, only the organic solvent is removed, leaving an aqueous suspension of nanoparticles. In some methods, rotary vacuum evaporation at a pressure of about 50 - 250 psi is used. In some methods, rotary vacuum evaporation at a pressure of about 200 psi for about 10 minutes is used to remove the organic solvent. In some methods, the emulsion from the high-pressure homogenizer may become clear after rotary vacuum evaporation, indicating that the organic solvent has been removed.

[0092] The material obtained after removal of the organic solvent can be subjected to centrifugation to recover the nanoparticles. In some methods, a two-step centrifugation process can be used. In a typical two-step centrifugation process, the obtained material can first be centrifuged at 3,000 - 5,000×g for 2 - 10 minutes. In some embodiments, the material obtained after solvent removal can be centrifuged at 4,400×g for about 3 minutes. After the first centrifugation step, the supernatant is collected and can be centrifuged at 20,000 - 25,000×g for 10 - 30 minutes using a high-speed centrifuge such as an Avanti® J-E high-speed centrifuge. In some methods, after the first centrifugation step, the supernatant is collected and can be centrifuged at about 22,000×g for about 15 minutes using a high-speed centrifuge. The pelleted nanoparticles can be resuspended in Milli-Q® IQ water (produced using a Milli-Q® IQ water purification system available from MilliporeSigma, St. Louis, MO).

[0093] In some embodiments, the polypeptide (e.g., IgY) in concentrated form is encapsulated within the nanoparticles. For example, in some embodiments, the polypeptide (e.g., IgY) is purified or partially purified and concentrated prior to nanoparticle formation. Methods for purifying and concentrating IgY antibodies from egg products are known in the art and are described, for example, in U.S. Patent No. 5,367,054 (Lee, 1994), which is hereby incorporated by reference in its entirety.

[0094] In certain embodiments, the nanoparticle composition containing the nanoparticles disclosed herein can be administered to a subject by oral administration. Eggs and egg yolks are natural food materials, are non-toxic, and are safe for oral ingestion. Dosage forms containing the nanoparticles provided herein, or compositions containing nanoparticles, may further include a pharmaceutically acceptable carrier suitable for oral administration. In some embodiments, the pharmaceutically acceptable carrier includes compounds generally recognized as safe (GRAS) by the FDA. In some embodiments, the GRAS compounds are acetic acid, aconitic acid, adipic acid, alginic acid, an α-amylase enzyme preparation derived from Bacillus stearothermophilus, benzoic acid, bromelain, caprylic acid, a carbohydrase and protease enzyme mixed product, citric acid, catalase (bovine liver), lactic acid, enzymatically modified lecithin, linoleic acid, malic acid, potassium bitartrate, propionic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, tartaric acid, diacetyl tartrate esters of monoglycerides and diglycerides, agar, brown algae, red algae, ammonium alginate, ammonium bicarbonate, ammonium carbonate, ammonium chloride, ammonium hydroxide, dibasic ammonium citrate, ammonium phosphate (monobasic), ammonium phosphate (dibasic), ammonium sulfate, a carbohydrase enzyme preparation derived from bacteria, a protease enzyme preparation derived from bacteria, bentonite, benzoyl peroxide, n-butane and isobutane, calcium acetate, calcium alginate, calcium carbonate, calcium chloride, calcium citrate, calcium gluconate, calcium glycerophosphate, calcium hydroxide, calcium iodate, calcium lactate, calcium oxide, calcium pantothenate, calcium propionate, calcium stearate, calcium sulfate, carbon dioxide, beta-carotene, Trichoderma longibrachiatumCellulase enzyme preparation derived from longibrachiatum, clove and its derivatives, cocoa butter substitute, copper gluconate, copper sulfate, corn silk and corn silk extract, cuprous iodide, L-cysteine, L-cysteine monohydrochloride, dextrin, diacetyl, dill and its derivatives, enzyme-modified fat, ethyl alcohol, ethyl formate, ferric ammonium citrate, ferric chloride, ferric citrate, ferric phosphate, ferric pyrophosphate, ferric sulfate, ferrous ascorbate, ferrous carbonate, ferrous citrate, ferrous fumarate, ferrous gluconate, ferrous lactate, ferrous sulfate, fishin, garlic and its derivatives, glucono-delta-lactone, corn gluten, wheat gluten, glyceryl monooleate, glyceryl monostearate, glyceryl behenate, glyceryl palmitostearate, acacia (gum arabic), ghatti gum, guar gum, locust (carob) bean gum, karaya gum (sterculia gum), tragacanth gum, gellan gum, xanthan gum, hydrogen peroxide, inositol, insoluble glucose isomerase enzyme preparation, elemental iron, isopropyl citrate, lactase enzyme preparation derived from Candida pseudotropicalis, lactase enzyme preparation derived from Kluyveromyces lactis, lecithin, licorice and licorice derivatives, ground limestone, animal lipase, Rhizopus niveusLipase enzyme preparation derived from niveus, magnesium carbonate, magnesium chloride, magnesium hydroxide, magnesium oxide, magnesium phosphate, magnesium stearate, magnesium sulfate, malt, maltodextrin, malt syrup (malt extract), manganese chloride, manganese citrate, manganese gluconate, manganese sulfate, menhaden oil, methylparaben, microparticulated protein product, monoglycerides and diglycerides, monosodium phosphate derivatives of monoglycerides and diglycerides, niacin, niacinamide, nickel, niacin preparation, nitrogen, nitrous oxide, peptone, rapeseed oil, beef bile extract, ozone, pancreatin, papain, pectin, pepsin, potassium alginate, potassium bicarbonate, potassium carbonate, potassium chloride, potassium citrate, potassium hydroxide, potassium iodide, potassium iodate, potassium lactate, potassium sulfate, propane, propyl gallate, propylene glycol, propylparaben, pyridoxine hydrochloride, rennet (animal-derived) and chymosin preparation (fermentation-derived), riboflavin, riboflavin-5-phosphate (sodium), henna, henna oil, cyan nut oil, sodium acetate, sodium alginate, sodium benzoate, sodium bicarbonate, sodium carbonate, sodium citrate, sodium diacetate, sodium hydroxide, sodium hypophosphite, sodium lactate, sodium metasilicate, sodium propionate, sodium sesquicarbonate, sodium tartrate, sodium potassium tartrate, sodium thiosulfate, sorbitol, stannous chloride (anhydrous and dihydrate), starter distillate, stearyl citrate, sucrose, corn sugar, invert sugar, corn syrup, high fructose corn syrup, thiamine hydrochloride, thiamine mononitrate, α-tocopherol, triacetin, tributyrin, triethyl citrate, trypsin, urea, urease enzyme preparation derived from Lactobacillus fermentum, vitamin A, vitamin B12, vitamin D, beeswax (yellow and white), candelilla wax, carnauba wax, whey, low lactose whey, low mineral whey, whey protein concentrate, baker's yeast extract, brewer's yeast extract, zein, and Lactococcus lactisIt is selected from aminopeptidase enzyme preparations derived from Lactococcus lactis, or any combination thereof.

[0095] In certain embodiments, the dosage form containing the nanoparticles or the composition containing the nanoparticles provided herein may include one or more additional compounds, such as nutrients or probiotics. For example, in one embodiment, the nanoparticles or the composition containing the nanoparticles can be incorporated into a dietary supplement.

[0096] One method of preparing the highly immunized eggs to be incorporated into the nanoparticles involves drying the eggs into egg powder. Various methods for drying eggs are known, including freeze-drying and spray-drying. In one method, spray-drying is used. The process of spray-drying eggs is well-known in the art.

[0097] In certain embodiments, whole eggs are divided into separate fractions, such as egg yolk and egg white. For example, it is generally known in the art that IgY antibodies are found in egg yolk. Thus, one of ordinary skill in the art will clearly recognize that separation of the egg yolk can provide a more effective fraction or remove unwanted components. Such further separation provides the ability to produce nanoparticles containing a more effective egg fraction. In some embodiments, IgY is separated from the highly immunized egg product and included in the nanoparticles. The separated IgY can be processed into a freeze-dried powder or a spray-dried powder to be included in the nanoparticles. IgY can be further purified before being incorporated into the nanoparticles.

[0098] In certain embodiments, the present disclosure relates to a method of delivering a polypeptide (e.g., IgY) to a subject, the method comprising administering to the subject, by oral administration, the nanoparticles or the composition containing the nanoparticles described herein. The nanoparticles or the composition containing the nanoparticles are preferably administered to the subject in an amount effective to treat or prevent a particular disorder. The dosage and the administration period depend on the particular condition of the subject. In some embodiments, the nanoparticles or the composition containing the nanoparticles are administered to the subject for at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 60, 90, 180 or 365 days. The nanoparticles or the composition containing the nanoparticles can be administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more per day. Any of these values can be used to define the range of the number of times the composition can be administered to the subject per day. For example, in some embodiments, the nanoparticles or the composition containing the nanoparticles are administered to the subject 1 to 2 times per day, 1 to 3 times per day, or 1 to 4 times per day. In some embodiments, the nanoparticles or the composition containing the nanoparticles are administered to the subject at least 2 times per day. In some embodiments, the nanoparticles or the composition containing the nanoparticles are administered to the subject at least 1 time per day.

[0099] In some embodiments, the composition is administered to the subject daily. In some embodiments, the nanoparticles or the composition containing the nanoparticles are administered to the subject once every two days. In some embodiments, the nanoparticles or the composition containing the nanoparticles are administered to the subject once every three days. In some embodiments, the nanoparticles or the composition containing the nanoparticles are administered to the subject once a week. In certain embodiments, the nanoparticles or the composition containing the nanoparticles are administered to the subject once a day for more than 10 consecutive days. Also provided is the use of the nanoparticles or the composition containing the nanoparticles described herein for delivering a polypeptide to a subject.

[0100] In some embodiments, a daily dose having an equivalent amount of highly immunized whole eggs in the range of 1 to less than several (or a highly immunized egg product containing an equivalent amount of 1 to less than several highly immunized whole eggs) can be administered to a subject according to a specific situation of the condition.

[0101] In certain embodiments, an effective amount of a highly immunized egg product in the form of nanoparticles administered to a subject (e.g., a human) is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 grams per day. For example, in some embodiments, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 grams of whole egg nanoparticles per day are administered to the subject. In some embodiments, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 grams of egg yolk nanoparticles per day are administered to the subject. In some embodiments, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 grams of dried egg yolk or dried whole egg nanoparticles per day are administered to the subject. Any of these values can be used to define the range of the effective amount of nanoparticles containing a highly immunized egg product administered to a mammal. For example, in some embodiments, the effective amount of nanoparticles containing a highly immunized egg product is between 0.1 and 10 grams per day, between 0.5 and 6 grams per day, or between 1 and 5 grams per day. In certain embodiments, an amount of nanoparticles containing IgY corresponding to the amount of IgY in 3 grams of egg yolk is administered to a subject (e.g., a human) per day.

Example

[0102] Example 1. IgY Quantification Using NanoDrop (trademark) One IgY and serial dilutions of IgY in the range of 0.105 to 26.9 mg / mL were analyzed for A280 (absorbance at 280 nm) values using a NanoDrop™ One Microvolume UV-Vis Spectrophotometer (Thermo Scientific). The sample type was selected as IgY, and baseline correction was performed at 320 nm. 2 μL of 1× phosphate buffered saline (PBS) was placed on the lower pedestal and lower arm for blanking (auto blank was on). The samples were gently vortexed before measurement. 2 μL aliquots of each sample were loaded and the respective A280 values were recorded. All sample dilutions were performed using 1× PBS. The A280 values are shown in Table 1.

[0103]

Table 1

[0104] The results are shown graphically in Figure 1, which provides a calibration curve for IgY quantification. The R 2 value was determined to be 0.9999.

[0105] Example 2. Preparation of IgY nanoparticles Nanoparticles containing IgY antibodies were prepared. To achieve efficient loading of IgY into the nanoparticles, a high-pressure homogenizer (HPH) was utilized. The high-pressure homogenization process facilitates the exposure of the formulation to multiple forces such as shear, cavitation, turbulent flow, impact force, and high pressure, which aids in the achievement of nanoparticles with optimal characteristics such as minimum particle size and maximum drug loading. EUDRAGIT® L 100 was used in the preparation of IgY nanoparticles, either alone or in combination with EUDRAGIT® S 100. An aqueous phase containing an IgY solution corresponding to 20 mg of IgY and 1% polyvinyl alcohol (PVA) was prepared. PVA was used as a surfactant polymer. An organic phase containing 3 mL of dichloromethane (DCM) and 1 mL of absolute ethanol was prepared. The EUDRAGIT® polymer was dissolved in absolute ethanol using an ultrasonic bath. The solution containing IgY and PVA, the solution containing DCM, and the alcohol solution of the EUDRAGIT® polymer were subjected to probe homogenization at 10,000 rpm for 1 minute using a digital high-speed homogenizer (Fisherbrand™ 850 Homogenizer, Fisher Scientific, Waltham, MA). The resulting crude emulsion was processed through an HPH (NanoDeBee homogenizer, BEE International, San Diego, CA). The detailed HPH parameters used for homogenization were as follows: nozzle type: N5; pressure: 15,000 psi; flow pattern: reverse; number of reactors: 6; number of cycles: 7. During high-pressure homogenization, Milli-Q® water (generated using a Milli-Q® IQ water purification system available from MilliporeSigma, St. Louis, MO) was added to collect 45 mL of the preparation. The formulation was then subjected to rotary vacuum evaporation at approximately 200 psi for approximately 10 minutes to remove the organic solvent. The end point was determined when the solution became clear. The resulting solution was centrifuged at 4,400×g for 3 minutes.The supernatant was collected and centrifuged at 22,000×g for 15 minutes using an Avanti® J-E high-speed centrifuge (Beckman Coulter Life Sciences, Indianapolis, IN). The collected nanoparticles were resuspended in 1 mL of Milli-Q® water.

[0106] Example 3. Physicochemical Characterization of IgY Nanoparticles IgY nanoparticles were analyzed for their physicochemical characteristics. The IgY nanoparticles were frozen at -80 °C overnight. Then, the frozen IgY nanoparticles were subjected to freeze-drying for 24 hours using an L Benchtop Freeze Dry system. The freeze-dried IgY nanoparticle powder was collected, and a known amount was dissolved in pH 7.4 buffer and then analyzed for IgY content by NanoDrop™ One analysis.

[0107] 10 μL of the nanoparticle formulation was diluted with 2 mL of Milli-Q® water and sonicated for 30 minutes. The particle size, polydispersity index (PDI), and zeta potential were measured using a Malvern zetasizer. The encapsulation efficiency % (%EE) of the nanoparticles loaded with IgY was also determined.

[0108] The IgY nanoparticles formulated with EUDRAGIT® L 100 (IgY-L nanoparticles) were found to have an average particle size of 276.0 nm and a polydispersity index (PDI) of 0.22. The IgY nanoparticles formulated with EUDRAGIT® L 100 / S 100 (IgY-L:S nanoparticles, the ratio of L100:S100 was 4:1) were found to have an average particle size of 272.1 nm and a polydispersity index (PDI) of 0.17. These data suggest that the IgY nanoparticles have a uniform particle size distribution.

[0109] The zeta potential of IgY-L nanoparticles was found to be -38.9 mV, and the zeta potential of IgY-L:S nanoparticles was found to be -31.2 mV. Therefore, the nanoparticles were anionic. Since cationic nanoparticles can cause toxicity and cell death, anionic nanoparticles have higher stability during storage and higher safety during administration to humans or animals.

[0110] IgY was effectively encapsulated into the nanoparticles, achieving encapsulation efficiency (EE)% of 35.03 ± 3.8% for IgY-L nanoparticles and 36.4 ± 1.99% for IgY-L:S nanoparticles. Encapsulation efficiency is the percentage of IgY in the sample encapsulated in the nanoparticles.

[0111] Example 4. ELISA assay Functional ELISA assays were performed using IgY extracted from nanoparticles and compared with control IgY at various concentrations. Nanoparticle formulations loaded with IgY (IgY-L and IgY-L:S nanoparticles) were lysed, and the lysed samples were diluted to obtain a concentration of 300 ng / mL for IgY. The diluted samples were then quantified for IgY amount using an ELISA assay. Briefly, the antigen was diluted to 1 mg / mL in bicarbonate buffer pH 9.0. 100 μL of this antigen solution was added to the ELISA plate wells in a 96-well plate. The plate was then covered with parafilm and incubated overnight at 4 °C. The solution was then discarded by tipping / ficking the plate, followed by washing it once with 200 μL of PBS (PBST) containing 0.05 wt% Tween-20. 200 μL of fresh blocking reagent (1% BSA in 1× PBS) was then added to each well and incubated for 1 hour at 37 °C. Next, the plate was washed once with PBST for 5 minutes. It was then tipped on a paper towel and tapped gently. The primary antibody from the stock solution diluted in PBST was added. Serial dilutions of IgY were performed in the range of 37 - 300 ng / mL, and the plate was incubated for 1 hour at 37 °C. The plate was then washed 3 times with PBST, each wash being for 5 minutes with 100 μL of PBST without agitation. The secondary antibody diluted in the blocking solution was added, incubated for 1 hour at 37 °C, followed by washing 2 times with PBST and finally once with PBS only, each wash being for 5 minutes. 100 μL of the chromogenic solution (TMB substrate solution) was then added and incubated at room temperature for 15 - 20 minutes, followed by the addition of 100 μL of the stop solution. The plate was read for absorbance values at 450 nm using a plate reader.

[0112] For the quantification of IgY and its serial dilutions using a functional ELISA assay, calibration on the plot was constructed. The obtained equation was y = 0.0058x + 1.8906. The absorbance values obtained from the lysed samples of IgY nanoparticles were incorporated into the above equation to calculate the amount of IgY. From the results, it was determined that IgY-L nanoparticles contained 274.2 ± 6.7 ng / mL of IgY antibody and IgY-L:S nanoparticles contained 273.8 ± 6.4 ng / mL of IgY antibody. Regarding the loading percentage of nanoparticles, IgY-L nanoparticles contained 5.84% (w / w) ± 0.64 of IgY per weight of nanoparticles, and IgY-L:S nanoparticles contained 6.07% (w / w) ± 0.33 of IgY per weight of nanoparticles.

[0113] Example 5. Preparation of Capsules Loaded with IgY Nanoparticles Hard gelatin capsules of size #3 were manually filled with lyophilized IgY nanoparticles. The loaded capsules were coated with either coating formulation A or B. The composition of coating formulation A is shown in Table 2. The composition of coating formulation B is shown in Table 3.

[0114] [Table 2]

[0115] [Table 3]

[0116] The actual amounts of the components for formulation can be appropriately scaled up according to the batch size to be manufactured. The required amounts of EUDRAGIT® L100 (0.6 g) [Formulation A] or EUDRAGIT® L100 (0.048 g), EUDRAGIT® S 100 (0.012 g) [Formulation B], talc (0.3 g), and triethyl citrate (0.06 g) were weighed. The required amounts of solvents: isopropyl alcohol (5.1 mL), milli-Q water (0.42 mL), and acetone (3.4 mL) were measured. All the weighed components were added to the mixture of the aforementioned solvents and subjected to an ultrasonic bath for 30 minutes. The resulting coating composition was transferred to a 25 mL glass beaker. Subsequently, the capsules containing IgY nanoparticles were immersed in the coating formulation. The immersion coating was performed 3 times with a 5-minute drying step between coatings. Then, the capsules were dried using a dryer with a hot air stream. Subsequently, the integrity of the coated capsules was confirmed, and it was determined that the coating process did not affect the integrity of the capsules.

[0117] Example 6. In Vitro Release Study Release studies of the coated blank capsules and the coated capsules containing IgY (Formulation A: EUDRAGIT® L100, and Formulation B: EUDRAGIT® L 100 and S 100) were performed in buffer solutions (50 mL volume) of pH 1.2 and pH 7.0 and carried out at 37 °C. The coated capsules loaded with IgY nanoparticles were placed in the release medium (50 mL) using a sinker, and 1 mL of the sample was collected at predetermined time points (pH 1.2: 1 and 2 hours, pH 7.0: 1 hour). After 2 hours at pH 1.2, the buffer solution was removed and replaced with the buffer solution of pH 7.0. The collected samples were centrifuged at 30,000 rpm for 15 minutes and subsequently filtered using a 0.2 μm syringe filter. The filtrate was analyzed using a NanoDrop™ One spectrophotometer to determine the amount of IgY released from the capsules.

[0118] Capsules containing IgY nanoparticles and coated with coating formulation A were found to release approximately 46.3% of IgY after 1 hour and 99.0% of IgY after 2 hours in a buffer at pH 1.2. After replacing the buffer at pH 1.2 with a buffer at pH 7.0, approximately 6.5% of IgY was released after 1 hour. Capsules containing IgY nanoparticles and coated with coating formulation B were found to release approximately 90.7% of IgY after 1 hour and 91.3% of IgY after 2 hours in a buffer at pH 1.2. After replacing the buffer at pH 1.2 with a buffer at pH 7.0, approximately 11.5% of IgY was released after 1 hour.

Claims

1. Nanoparticles comprising a composition containing a polypeptide, wherein the composition is encapsulated in a material comprising a biocompatible biodegradable polymer and a surfactant polymer.

2. The nanoparticles according to claim 1, wherein the biocompatible biodegradable polymer is polymethacrylate.

3. The nanoparticles according to claim 1, wherein the nanoparticles have a diameter of less than 300 nm.

4. The nanoparticles according to claim 1, wherein the nanoparticles contain at least 5% w / w of the polypeptide.

5. The nanoparticles according to any one of claims 1 to 4, wherein the polypeptide is IgY.

6. The nanoparticles according to claim 5, wherein the IgY is obtained from hyperimmunized eggs.

7. The nanoparticles according to any one of claims 1 to 4, wherein the composition is a hyperimmunized egg product.

8. The nanoparticles according to any one of claims 1 to 4, wherein the composition is a defatted fraction from egg yolk containing at least 95% by weight of IgY.

9. The nanoparticles according to any one of claims 1 to 4, wherein the composition contains purified IgY.

10. The biocompatible biodegradable polymer is (a) methacrylic acid-methyl methacrylate copolymer (1:1), or (b) methacrylic acid-methyl methacrylate copolymer (1:2), or (c) both (a) and (b) The nanoparticles according to any one of claims 1 to 9, comprising.

11. The nanoparticles according to any one of claims 1 to 10, wherein the surfactant polymer is selected from chitosan, poly(vinyl) alcohol (PVA), polyvinylpyrrolidone (PVP), and combinations thereof.

12. The nanoparticles according to any one of claims 1 to 11, and a pharmaceutically acceptable carrier A pharmaceutical composition comprising.

13. The pharmaceutical composition according to claim 12, wherein the pharmaceutically acceptable carrier is suitable for oral administration.

14. The pharmaceutical composition according to claim 12 or 13, wherein the pharmaceutical composition is in a form selected from the group consisting of powders, tablets, enteric-coated tablets, capsules, enteric-coated capsules, suspensions, solutions, and oral beverages.

15. A method for delivering a polypeptide, comprising: administering the nanoparticles according to any one of claims 1 to 11 to a subject by oral administration, or Administering the pharmaceutical composition according to any one of claims 12 to 14 to a subject by oral administration A method comprising:

16. The method according to claim 15, wherein the polypeptide contained in the nanoparticles is IgY.

17. The method according to claim 16, wherein the IgY is obtained from hyperimmunized eggs.

18. The method according to claim 15, wherein the composition contained in the nanoparticles is a hyperimmunized egg product.

19. The method according to claim 15, wherein the composition contained in the nanoparticles is a defatted fraction from egg yolk containing at least 95% by weight of IgY.

20. The method according to claim 15, wherein the composition contained in the nanoparticles contains purified IgY.

21. The method according to any one of claims 15 to 20, wherein the biocompatible and biodegradable polymer of the nanoparticles contains polymethacrylate.

22. The method according to any one of claims 15 to 21, wherein the nanoparticles have a diameter of less than 300 nm.

23. The method according to any one of claims 15 to 22, wherein the nanoparticles contain at least 5% w / w of the polypeptide.

24. (a) Preparing a first mixture comprising a composition containing a polypeptide, water, and a surfactant polymer; (b) Sonication of the first mixture; (c) Adding a biocompatible and biodegradable polymer to ethanol and sonicating to produce a second mixture; (d) Mixing the first mixture and the second mixture with an organic solvent to form a third mixture; (e) Homogenizing the third mixture at a speed of about 5,000 to 15,000 rpm using a probe homogenizer to produce a coarse emulsion; (f) Subjecting the coarse emulsion to high-pressure homogenization at a pressure of about 10,000 psi to 30,000 psi using a high-pressure homogenizer to form a fourth mixture; and (g) Evaporating ethanol and the organic solvent from the fourth mixture to produce a suspension containing nanoparticles A method for preparing the nanoparticles according to claim 1, comprising:

25. The method according to claim 24, further comprising centrifuging the suspension to obtain the nanoparticles.

26. The method according to claim 24 or 25, wherein the surfactant polymer is selected from chitosan, poly(vinyl) alcohol (PVA), polyvinylpyrrolidone (PVP), and combinations thereof.

27. The method according to any one of claims 24 to 26, wherein the organic solvent contains dichloromethane.

28. The method according to any one of claims 24 to 27, wherein the first mixture contains about 2% to 5% (w / w) of the polypeptide.

29. The method according to any one of claims 24 to 28, wherein the polypeptide is IgY.

30. The method according to any one of claims 24 to 29, wherein the second mixture contains a biocompatible and biodegradable polymer at 10 mg / mL to 20 mg / mL.

31. (a) The surfactant polymer contains 0.1% (w / w) of chitosan, or (b) The surfactant polymer contains 0.5 to 2.5% (w / w) of poly(vinyl) alcohol (PVA), or (c) The surfactant polymer contains 0.5 to 2.5% (w / w) of polyvinylpyrrolidone (PVP), or (d) It is any combination of (a) to (c), The method according to any one of claims 24 to 30.

32. The method according to any one of claims 24 to 31, wherein the first mixture contains 0.75% to 2% (w / w) of poly(vinyl) alcohol (PVA).

33. Use of the nanoparticles according to any one of claims 1 to 11 for delivering a polypeptide to a subject.

34. Use of the pharmaceutical composition according to any one of claims 12 to 14 for delivering a polypeptide to a subject.

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