Formulations for oral immunotherapy against eggs, methods of manufacture, and treatment of egg allergy

Abstract

Described herein are egg white protein preparations, dosage containers containing egg white protein preparations, methods for making the egg white protein preparations and dosage containers, methods for controlling the quality of the egg white protein preparations, materials for producing egg white protein preparations (such as dried egg white protein powder), and dosage containers. Further described herein is an oral immunotherapy for treating egg allergy. The method includes orally administering to a patient doses of a pharmaceutical composition comprising egg white protein according to an oral immunotherapy schedule. Also described herein are methods for adjusting the dosage of a pharmaceutical composition during oral immunotherapy if the patient experiences adverse events related to the administration of the dose or concurrent factors related to increased sensitivity to an allergen unrelated to the administration of the dose.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]

[0001] This application claims priority to U.S. Provisional Application No. 62 / 780,853, filed December 17, 2018, and U.S. Provisional Application No. 62 / 780,862, filed December 17, 2018. The contents of each of the above U.S. provisional applications are incorporated herein by reference in their entirety. [Technical field]

[0002]

[0002] The present invention relates to formulations for oral immunotherapy against eggs and methods for producing such formulations, as well as the oral immunotherapeutic treatment of egg allergy in patients. [Background technology]

[0003]

[0003] IgE-mediated hen egg allergy is one of the most common food allergies in childhood and can be associated with severe immediate hypersensitivity reactions, including life-threatening anaphylaxis. The prevalence of egg allergy is estimated to be about 0.5%-2.5% in Western countries. Most children typically outgrow their egg allergy over time, but cross-sectional studies indicate that overgrowth may not occur by the teenage years, and egg allergy persists in about 20% of individuals at age 18.

[0004]

[0004] Two major egg allergy phenotypes are recognized in the clinic: (1) allergy to both raw egg white and baked whole egg, and (2) allergy to raw egg white only. Approximately 20%-30% of egg-allergic individuals are allergic to both raw egg white and baked whole egg, and 70%-80% of egg-allergic individuals are allergic to raw egg white only.

[0005]

[0005] The current standard of care for management of egg allergy is dietary avoidance of eggs and patient / family education regarding recognition and management of allergic symptoms and appropriate use of rescue medications (e.g., epinephrine autoinjectors (EAIs)). However, avoiding eggs is extremely difficult for egg-allergic patients due to the ubiquity of egg as an ingredient in many food products and its importance as a childhood nutrient. The burden of avoiding and constantly fearing accidental exposure can adversely affect the health-related quality of life of egg-allergic individuals and their families. According to the medical need, daily carry and emergency use of EAIs for the treatment of anaphylaxis appears to be insufficient, which can result in adverse outcomes including hospitalization and death.

[0006]

[0006] It is generally believed that ingestion of as little as 1 milligram of egg protein can induce an allergic reaction, based on studies with estimated population thresholds. Although the threshold level at which an allergic reaction is induced varies widely across egg-allergic populations, the ED10 (the elicitation dose predicted to cause a reaction in 10% of individuals with a particular food allergy) is estimated to be in the range of 3.7 mg to 5.8 mg of egg protein. As a result, occasional allergic reactions to eggs are common.

[0007]

[0007] Currently available therapies are designed to treat only the symptoms of allergic reactions when they occur. Therefore, in the absence of treatments, therapies that have the potential to reduce the risk of severe allergic reactions in the event of accidental exposure represent an urgent unmet medical need. However, the development of carefully calibrated dosage forms for delivering egg allergens by oral immunotherapy remains a challenge, especially for low-dose forms. At low doses, the control of allergenic materials in oral immunotherapy administration is particularly important to limit treatment-related adverse events. Therefore, bulk manufacturing processes, especially when producing low doses, should have sufficient uniformity throughout the mixed product to ensure sufficient dose uniformity in packaged products.

[0008]

[0008] Recently, oral immunotherapy (OIT) of egg allergy has been investigated, which involves orally administering increasing doses of allergenic proteins to patients to achieve a state of desensitization. However, further improvements in the safety and / or efficacy of the treatment are desirable. Summary of the Invention [Means for solving the problem]

[0009]

[0009] Described herein are egg white protein preparations, dosage forms containing the egg white protein preparations, and methods of making such egg white protein preparations and dosage forms.

[0010]

[0010] In some embodiments, a method of making an egg white protein preparation includes: (a) mixing dry egg white protein powder with a first amount of a first diluent to form a first mixture; (b) passing the first mixture through a mesh screen; (c) after steps (a) and (b), mixing the first mixture with a second amount of the first diluent to form a second mixture; (d) mixing the second mixture with a second diluent to form a third mixture; (e) mixing the third mixture at a higher shear force than that used for mixing in step (c); and (f) mixing the third mixture with a lubricant to form the egg white protein preparation. In some embodiments, the egg white protein preparation has about 0.05% to about 2.5% by weight of egg white protein. In some embodiments, the egg white protein preparation has about 0.1% to about 0.7% by weight of egg white protein. In some embodiments, step (b) includes passing at least a portion of the second amount of the first diluent through a mesh screen with the first mixture. In some embodiments, step (b) includes passing the second amount of the first diluent through a mesh screen with the first mixture. In some embodiments, step (c) includes multiple substeps, each substep including (i) adding a portion of the second amount of the first diluent to the first mixture, and (ii) mixing a portion of the second amount of the first diluent and the first mixture. In some embodiments, step (c) includes three or more substeps. In some embodiments, step (c) includes continuously mixing the first mixture with the second amount of the first diluent as the second amount of the first diluent is added to the first mixture. In some embodiments, the first mixture is mixed with the second amount of the first diluent in a tumble blender. In some embodiments, the second mixture is mixed with the second diluent in a tumble blender. In some embodiments, the third mixture is mixed using a conical mill. In some embodiments, the third mixture is mixed with a lubricant in a tumble blender. In some embodiments, a third amount of the first diluent is mixed with the third mixture.In some embodiments, a third amount of the first diluent and the lubricant are co-mixed with the third mixture. In some embodiments, step (f) comprises (i) passing the third mixture and the lubricant together through a mesh screen, and (ii) mixing the third mixture with the lubricant to form the egg white protein formulation. In some embodiments, the egg white protein formulation is substantially free of colloidal silicon dioxide. In some embodiments, the egg white protein formulation is about 40% to about 70% by weight of the first diluent. In some embodiments, the egg white protein formulation is about 30% to about 50% by weight of the second diluent. In some embodiments, the egg white protein formulation is about 0.1% to about 2% by weight of the lubricant.

[0011] In some embodiments, a method of making an egg white protein preparation includes: (a) mixing a dry egg white protein powder with a first amount of a first diluent to form a first mixture; (b) mixing a second amount of the first diluent with the first mixture to form a second mixture; (c) mixing the second mixture with a second diluent to form a third mixture; (d) mixing the third mixture at a higher shear force than that used for mixing in step (b); and (e) mixing the third mixture with a lubricant to form the egg white protein preparation. In some embodiments, the dry egg white protein powder is mixed with the first amount of the first diluent in step (a) at a higher shear force than that used for mixing in step (b). In some embodiments, the egg white protein preparation has about 1% to about 70% by weight of egg white protein. In some embodiments, the dried egg white protein powder is mixed with a first amount of the first diluent in step (a) using a conical mill. In some embodiments, step (b) includes two mixing substeps, one mixing substep being at a higher shear than the other mixing substep. In some embodiments, step (c) includes mixing the second mixture with an additional amount of the first diluent before mixing with the second diluent to form a third mixture. In some embodiments, step (c) further includes mixing the second mixture together with the first diluent and additional amounts of the second diluent to form a third mixture. In some embodiments, the third mixture is mixed in step (d) using a conical mill. In some embodiments, the second amount of the first diluent and the first mixture are mixed in a tumble blender. In some embodiments, the second mixture is mixed with the second diluent in a tumble blender. In some embodiments, a third amount of the first diluent is mixed with the third mixture. In some embodiments, a third amount of the first diluent and a lubricant are combined and mixed into the third mixture.In some embodiments, step (e) comprises: (i) mixing a portion of the third mixture with a lubricant; (ii) passing the mixture of (i) through a mesh screen; and (iii) mixing the mixture of (ii) with an additional portion of the third mixture to form the egg white protein formulation. In some embodiments, the third mixture is mixed with the lubricant in a tumble blender. In some embodiments, the egg white protein formulation is about 9% to about 85% by weight of the first diluent. In some embodiments, the egg white protein formulation is about 10% to about 50% by weight of the second diluent. In some embodiments, the egg white protein formulation is about 10% to about 20% by weight of the second diluent. In some embodiments, the egg white protein formulation is about 0.1% to about 2% by weight of the lubricant. In some embodiments, the egg white protein formulation comprises a lubricant. In some embodiments, the lubricant is mixed with the egg white protein powder and the first amount of the first diluent during or before step (a). In some embodiments, the lubricant is colloidal silicon dioxide. In some embodiments, the egg white protein formulation is about 2% to about 70% by weight of egg white protein. In some embodiments, the egg white protein formulation is substantially free of colloidal silicon dioxide and may contain about 1% to about 5% by weight of egg white protein.

[0012] In some embodiments, a method of making an egg white protein preparation includes: (a) mixing dry egg white protein powder with a first diluent and a lubricant to form a first mixture; (b) mixing a second diluent and the first mixture at a higher shear than that used in mixing in step (a) to form a second mixture; and (c) mixing the second mixture and the lubricant to form an egg white protein preparation. In some embodiments, the egg white protein preparation has about 50% to about 80% egg white protein by weight. In some embodiments, the dry egg white protein, the first diluent, and the lubricant are mixed in a tumble blender. In some embodiments, the second diluent and the first mixture are mixed in step (b) using a conical mill. In some embodiments, the second mixture and the lubricant are mixed in a tumble blender. In some embodiments, a second amount of the first diluent is mixed with the second mixture. In some embodiments, a second amount of the first diluent and a lubricant are combined and mixed with the second mixture. In some embodiments, the lubricant comprises colloidal silicon dioxide. In some embodiments, step (c) comprises (i) mixing a portion of the second mixture and the lubricant, (ii) passing the portion of the second mixture and the lubricant through a mesh screen, and (iii) mixing the mixture of (ii) with an additional portion of the second mixture to form the egg white protein formulation.

[0013] In some embodiments of the above method, the egg white protein preparation is produced in a lot size of about 5 kg or more. In some embodiments, the egg white protein preparation is produced in a lot size of about 5 kg to about 50 kg.

[0014] In some embodiments of the above method, the method further comprises measuring an egg white protein blend uniformity of the egg white protein preparation. In some embodiments, the egg white protein preparation has an egg white protein blend uniformity relative standard deviation (RSD) of about 15% or less.

[0015] In some embodiments of the above method, the method further includes packaging the egg white protein preparation into a plurality of dosage containers. In some embodiments, the dosage container is a capsule or sachet. In some embodiments, the method further includes measuring the egg white protein content uniformity of the plurality of dosage containers. In some embodiments, the plurality of dosage containers have an egg white protein content uniformity relative standard deviation (RDS) of about 15% or less.

[0016]

[0016] In some embodiments of the above method, the first diluent is pregelatinized starch.

[0017]

[0017] In some embodiments of the above method, the second diluent is microcrystalline cellulose.

[0018]

[0018] In some embodiments of the above method, the lubricant is magnesium stearate.

[0019]

[0019] In some embodiments of the above method, the dry egg white protein powder contains about 50% to about 90% egg white protein by weight.

[0020]

[0020] In some embodiments of the above method, forming the dried egg white protein powder comprises spray drying liquid egg white.

[0021]

[0021] In some embodiments of the above method, the egg white protein powder is deglucose depleted.

[0022]

[0022] In some embodiments of the above method, the dried egg white powder is pasteurized.

[0023]

[0023] In some embodiments of the above method, the dried egg white protein powder is derived from chicken eggs.

[0024]

[0024] In some embodiments of the above method, the method further includes characterizing the ovomucoid, ovalbumin, ovotransferrin, or lysozyme in the dried egg white protein powder.

[0025]

[0025] In some embodiments of the above method, the method further includes characterizing ovomucoid, ovalbumin, ovotransferrin, or lysozyme in the egg white protein preparation.

[0026] In some embodiments, characterizing ovomucoid, ovalbumin, ovotransferrin, or lysozyme comprises obtaining a high performance liquid chromatography (HPLC) profile. In some embodiments, the HPLC profile is a reverse phase HPLC (RP-HPLC) profile. In some embodiments, the HPLC profile is a size exclusion chromatography HPLC (SEC-HPLC) profile. In some embodiments, the method comprises comparing the obtained HPLC profile to a reference HPLC profile.

[0027] In some embodiments, characterizing the ovomucoid, ovalbumin, ovotransferrin, or lysozyme comprises quantifying the amount of ovomucoid, ovalbumin, ovotransferrin, or lysozyme. In some embodiments, quantifying the amount of ovomucoid, ovalbumin, ovotransferrin, or lysozyme comprises measuring the amount of ovomucoid, ovalbumin, ovotransferrin, or lysozyme relative to total protein in the egg white protein powder or egg white protein formulation. In some embodiments, quantifying the amount of ovomucoid, ovalbumin, ovotransferrin, or lysozyme comprises measuring the amount of ovomucoid, ovalbumin, ovotransferrin, or lysozyme relative to the total amount of ovomucoid, ovalbumin, ovotransferrin, and lysozyme in the egg white protein powder or egg white protein formulation.

[0028] In some embodiments, characterizing ovomucoid, ovalbumin, ovotransferrin, or lysozyme comprises measuring the potency of ovomucoid, ovalbumin, ovotransferrin, or lysozyme in an egg white protein powder or an egg white protein formulation. In some embodiments, the potency of ovomucoid, ovalbumin, ovotransferrin, or lysozyme in an egg white protein powder or an egg white protein formulation is measured relative to the potency of ovomucoid, ovalbumin, ovotransferrin, or lysozyme in a reference sample. In some embodiments, the potency of ovomucoid, ovalbumin, ovotransferrin, or lysozyme is measured using an immunoassay. In some embodiments, the immunoassay comprises the use of an antibody that specifically binds ovomucoid, ovalbumin, ovotransferrin, or lysozyme. In some embodiments, the immunoassay comprises the use of a pool of antibodies comprising two or more antibodies selected from the group consisting of antibodies that specifically bind to ovomucoid, antibodies that specifically bind to ovalbumin, antibodies that specifically bind to ovotransferrin, or antibodies that specifically bind to lysozyme. In some embodiments, the antibodies are IgE or IgG antibodies. In some embodiments, the potency of ovomucoid, ovalbumin, ovotransferrin, or lysozyme is measured using an enzyme-linked immunosorbent assay (ELISA).

[0029]

[0029] Further described herein is an egg white protein preparation made according to any one of the above methods.

[0030]

[0030] In some embodiments, the egg white protein formulation comprises dried egg white protein powder, a first diluent, a second diluent, and a lubricant, and the egg white protein formulation is substantially free of colloidal silicon dioxide. In some embodiments, the egg white protein formulation comprises about 0.1% to about 3.5% by weight of egg white protein. In some embodiments, the first diluent is pregelatinized starch. In some embodiments, the second diluent is microcrystalline cellulose. In some embodiments, the lubricant is magnesium stearate. In some embodiments, the egg white protein formulation consists essentially of egg white protein powder, a first diluent, a second diluent, and a lubricant.

[0031] Further described herein are methods for treating egg allergy in a patient, and methods for adjusting the dosage of a pharmaceutical composition comprising egg white protein during oral immunotherapy for egg allergy.

[0032]

[0032] In one embodiment, a method for treating egg allergy in a patient comprises orally administering to the patient multiple doses of a pharmaceutical composition comprising egg white protein according to an oral immunotherapy schedule, the oral immunotherapy schedule including: (a) an up-dosing phase comprising orally administering to the patient a series of ascending doses of about 1 mg to about 300 mg of egg white protein, where a given dose is administered to the patient for at least 2 weeks before the dose is up-dosed, the dose up-dosing period being about 20 weeks to about 44 weeks in length; and (b) a maintenance period comprising orally administering to the patient multiple maintenance doses comprising egg white protein, where the maintenance period is about 12 weeks or longer in length.

[0033] In some embodiments, the patient's serum level of egg white specific IgE (ew-IgE) is about 7 kU at the start of treatment. A In some embodiments, the patient's serum level of egg white specific IgE (ew-IgE) is about 5 kU / L or more at the start of treatment. A / L or more.

[0034] In some embodiments, the series of increasing doses administered to the patient during the dose escalation period includes at least a 1 mg dose of egg white protein and a 300 mg dose of egg white protein. In some embodiments, the series of increasing doses administered to the patient during the dose escalation period includes at least 10 different doses of egg white protein. In some embodiments, the series of increasing doses administered to the patient during the dose escalation period includes doses of about 1 mg, about 3 mg, about 6 mg, about 12 mg, about 20 mg, about 40 mg, about 80 mg, about 120 mg, about 160 mg, about 200 mg, about 240 mg, and about 300 mg of egg white protein.

[0035]

[0035] In some embodiments, the dose administered during the dose escalation period is increased only if the patient tolerates the previous dose.

[0036]

[0036] In some embodiments, the maximum dose administered to a patient during the dose escalation period is about 300 mg of egg white protein.

[0037] In some embodiments, the maintenance dose administered to the patient during the maintenance period is about 300 mg or more of egg white protein. In some embodiments, the maintenance dose administered to the patient during the maintenance period is about 300 mg of egg white protein.

[0038]

[0038] In some embodiments, a maintenance dose is administered to a patient only if the patient tolerates the maximum dose administered to the patient during the dose escalation period.

[0039]

[0039] In some embodiments, the patient tolerates a dose of about 600 mg of raw egg white protein at the end of the maintenance period. In some embodiments, the patient tolerates a dose of about 1000 mg of raw egg white protein at the end of the maintenance period. In some embodiments, the patient tolerates a dose of about 2000 mg of raw egg white protein at the end of the maintenance period. In some embodiments, the patient tolerates a cumulative dose of about 2000 mg of cooked egg white protein at the end of the maintenance period. In some embodiments, the patient tolerates a cumulative dose of about 2000 mg of baked egg white protein at the end of the maintenance period.

[0040]

[0040] In some embodiments, the patient is unable to tolerate a dose of about 300 mg of raw egg white protein prior to the initiation of treatment. In some embodiments, the patient is unable to tolerate a cumulative dose of about 2000 mg of cooked egg white protein prior to the initiation of treatment. In some embodiments, the patient tolerates a cumulative dose of about 2000 mg of baked egg white protein prior to the initiation of treatment. In some embodiments, the patient is unable to tolerate a cumulative dose of about 2000 mg of baked egg white protein prior to the initiation of treatment. In some embodiments, the patient tolerates a cumulative dose of about 2000 mg of baked egg white protein prior to the initiation of treatment.

[0041]

[0041] In some embodiments, the oral immunotherapy schedule includes an initial titration phase prior to a dose escalation period, the initial titration phase comprising orally administering to the patient a series of ascending doses of about 0.2 mg to about 2 mg of egg white protein per day, wherein a single dose of any given dose is administered to the patient, the doses being spaced at least 15 minutes apart.

[0042]

[0042] In some embodiments, the patient is treated according to the oral immunotherapy schedule only if the patient tolerates a dose of about 1.2 mg of raw egg white protein on the first day of treatment.

[0043] In some embodiments, the patient is about 4 years of age or older prior to the initiation of treatment. In some embodiments, the patient is about 4 years of age to about 26 years of age prior to the initiation of treatment.

[0044]

[0044] Also described herein is a method of adjusting a dosage of a pharmaceutical composition comprising egg white protein during oral immunotherapy of egg allergy in a subject, the oral immunotherapy comprising: (i) a dose escalation period comprising orally administering to the patient a series of ascending doses of egg white protein; and (ii) a maintenance period comprising orally administering to the patient a plurality of maintenance doses comprising egg white protein, the method comprising orally administering to the patient a first dose of the pharmaceutical composition; and orally administering to the patient a second dose of the pharmaceutical composition, wherein if the patient experiences an adverse event associated with administration of the first dose, the second dose is reduced, skipped, or at least a portion of the dose is delayed. In some embodiments, the second dose is split into a first portion and a second portion, the first portion is administered according to a predetermined administration schedule, and if the patient experiences an adverse event associated with administration of the first dose, the second portion is delayed relative to the predetermined administration schedule. In some embodiments, the second portion is delayed about 8 hours to about 12 hours after the first portion is administered. In some embodiments, if the patient experiences an adverse event associated with administration of the first dose, the second dose is skipped. In some embodiments, if the patient experiences an adverse event associated with administration of the first dose, the second dose is reduced relative to the first dose. In some embodiments, the subsequent dose of the pharmaceutical composition is reduced relative to the first dose for about one week or more before titrating the subsequent dose. In some embodiments, the subsequent dose of the pharmaceutical composition is reduced relative to the first dose for about one week to about two weeks before attempting to titrate the subsequent dose. In some embodiments, the subsequent dose of the pharmaceutical composition is reduced relative to the first dose for about one week to about two weeks before titrating the subsequent dose. In some embodiments, the adverse event associated with administration of the first dose is a mild allergenic adverse event. In some embodiments, the adverse event associated with administration of the first dose is a moderate allergenic adverse event or a severe allergenic adverse event. In some embodiments, the first dose and the second dose are administered to the patient during a dose escalation period of oral immunotherapy. In some embodiments, the first dose and the second dose are administered to the patient during a maintenance period of oral immunotherapy.

[0045] Also described herein is a method of adjusting the dosage of a pharmaceutical composition comprising egg white protein during oral immunotherapy of egg allergy in a subject, the oral immunotherapy comprising (i) a dose escalation period comprising orally administering to the patient a series of ascending doses of egg white protein, and (ii) a maintenance period comprising orally administering to the patient a plurality of maintenance doses comprising egg white protein, the method comprising orally administering to the patient a first dose of the pharmaceutical composition and orally administering to the patient a second dose of the pharmaceutical composition, the second dose being reduced or skipped if the patient experiences a concomitant factor associated with increased sensitivity to an allergen unrelated to administration of the first dose. In some embodiments, the concomitant factor associated with increased sensitivity to an allergen is an atopic disease flare, inflammation, illness, or menstruation. In some embodiments, the second dose is skipped if the patient experiences a concomitant factor associated with increased sensitivity to an allergen unrelated to administration of the first dose. In some embodiments, if the patient experiences a concomitant factor associated with increased sensitivity to the allergen unrelated to administration of the first dose, the second dose is reduced relative to the first dose. In some embodiments, the subsequent dose of the pharmaceutical composition is reduced relative to the first dose for about one week or more before titrating the subsequent dose. In some embodiments, the subsequent dose of the pharmaceutical composition is reduced relative to the first dose for about one week to about two weeks before attempting to titrate the subsequent dose. In some embodiments, the subsequent dose of the pharmaceutical composition is reduced relative to the first dose for about one week to about two weeks before titrating the subsequent dose. In some embodiments, the first dose and the second dose are administered to the patient during a dose escalation period of oral immunotherapy. In some embodiments, the first dose and the second dose are administered to the patient during a maintenance period of oral immunotherapy. In some embodiments, the concomitant factor associated with increased sensitivity to the allergen is unintentional exposure to a food to which the patient is allergic.

[0046]

[0046] In some embodiments of the above method, the egg white protein in the pharmaceutical composition is raw egg white protein.

[0047]

[0047] In some embodiments of the above methods, the pharmaceutical composition is mixed with a food vehicle prior to administration.

[0048]

[0048] Further provided in the present specification is a pharmaceutical composition for use in the above-mentioned method for treating egg allergy, the pharmaceutical composition comprising an egg white protein preparation prepared according to the above-mentioned method for making an egg white protein preparation.

[0049]

[0049] Further provided herein is a pharmaceutical composition for use in the manufacture of a medicament for the method of treating egg allergy in a patient, in accordance with the above-described method of treating egg allergy, the pharmaceutical composition comprising an egg white protein preparation prepared in accordance with the above-described method of making an egg white protein preparation. [Brief description of the drawings]

[0050] [Figure 1] An exemplary method of making an egg white protein preparation, particularly at low dosage levels, is provided. [Diagram 2] An exemplary method of making an egg white protein preparation, particularly at an intermediate dosage level, is provided. [Diagram 3] An exemplary method of making an egg white protein preparation, particularly at high dosage levels, is provided. [Figure 4] 1 shows an exemplary decision tree for adjusting dosage depending on the severity of adverse events associated with administration of egg white protein during oral immunotherapy. [Diagram 5] FIG. 1 shows a size-exclusion chromatography HPLC chromatogram of dried egg white protein powder, identifying ovalbumin, ovomucoid, ovotransferrin, and lysozyme. [Figure 6] 1 shows a reverse-phase HPLC chromatogram of dried egg white protein powder, identifying ovalbumin, ovomucoid, ovotransferrin, and lysozyme. [Figure 7]Shown is an SDS-PAGE gel of dried egg white protein powder (lanes 3-5; approximately 2 µg protein per well) and protein standards (ovomucoid, ovalbumin, lysozyme, and ovotransferrin; lanes 7-10). [Figure 8] Shown are immunoblots of dried egg white protein powder (lanes 3-5) and protein standards (ovomucoid, ovalbumin, lysozyme, and ovotransferrin; lanes 7-10) that were stained to identify ovalbumin, ovomucoid, ovotransferrin, and lysozyme. [Figure 9] FIG. 1 shows a schematic diagram of a clinical trial of a pharmaceutical composition containing egg white protein. [Figure 10] FIG. 1 shows a schematic diagram of a clinical trial of a pharmaceutical composition containing egg white protein. [Figure 11] 1 shows a reversed-phase HPLC chromatogram of dried egg white protein powder using the improved RP-HPLC method, showing the relative retention time peaks identifying ovomucoid, lysozyme, ovotransferrin, and ovalbumin. [Figure 12A] An exemplary method of making an egg white protein preparation, particularly at low dosage levels, is provided. [Figure 12B] Another exemplary method of making an egg white protein preparation, particularly at low dosage levels, is provided. [Figure 12C] Another exemplary method of making an egg white protein preparation, particularly at low dosage levels, is provided. [Figure 13A] An exemplary method of making an egg white protein preparation, particularly at an intermediate dosage level, is provided. [Figure 13B] Another exemplary method of making an egg white protein preparation, particularly at an intermediate dosage level, is provided. [Figure 13C] Another exemplary method of making an egg white protein preparation, particularly at an intermediate dosage level, is provided. [Figure 14A] An exemplary method of making an egg white protein preparation, particularly at high dosage levels, is provided. [Figure 14B] Another exemplary method of making an egg white protein preparation, particularly at high dosage levels, is provided. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051]

[0069] Described herein are egg white protein preparations and methods of making such egg white protein preparations. Egg white protein preparations include dried egg white protein powder along with one or more diluents, lubricants, and (in some formulations) glidants, and can be useful for treating egg allergy through oral immunotherapy. Because hyperallergic patients may be sensitive to slight variations in the amount of allergen contained in the formulation, it is desirable to carefully calibrate the amount and quality of egg allergen contained within the formulation, for example, by using the quality control and / or manufacturing processes described herein. The manufacturing methods described herein allow for the production of egg white protein preparations with carefully controlled amounts of egg allergen.

[0052]

[0070] A significant challenge in the production of egg white protein formulations is the scale-up of lot sizes. Research bench-top lots with less uniformity are more readily available than large-scale production for commercial production. Obtaining a sufficiently homogenous formulation blend is particularly challenging and important for low-dose formulations, as patients taking such doses may be particularly sensitive to egg white allergens. The manufacturing methods described herein provide for the scale-up of egg white protein formulation production, including batches larger than 5 kilograms.

[0053]

[0071] In some methods of making an egg white protein preparation, the method includes: (a) mixing a dry egg white protein powder with a first amount of a first diluent to form a first mixture; (b) passing the first mixture through a mesh screen; (c) after steps (a) and (b), mixing the first mixture with a second amount of the first diluent to form a second mixture; (d) mixing the second mixture with a second diluent to form a third mixture; (e) mixing the third mixture with a higher shear force than that used for mixing in step (c); and (f) mixing the third mixture with a lubricant to form the egg white protein preparation. In step (e), the third mixture is mixed to disaggregate particle aggregates. Step (c) may occur in multiple substeps to serially dilute the mixture with two or more different portions of the second amount of the first diluent. This method of preparation of the formulation is particularly useful for formulations used to prepare low dose containers containing formulations in doses of about 0.1 mg to about 12 mg. The formulations may have, for example, about 0.05% to about 2.5% by weight of egg white protein. A lubricant (e.g., colloidal silicon dioxide) need not be included in the low dose formulation.

[0054]

[0072] In some methods of making an egg white protein formulation, the method includes (a) mixing a dry egg white protein powder with a first amount of a first diluent to form a first mixture; (b) mixing a second amount of the first diluent with the first mixture to form a second mixture; (c) mixing the second mixture with a second diluent to form a third mixture; (d) mixing the third mixture at a shear force higher than that used for mixing in step (b); and (e) mixing the third mixture with a lubricant to form the egg white protein formulation. In step (d), the third mixture is mixed to disaggregate particle aggregates in the mixture. Step (a) may also be performed at a shear force higher than that used for mixing in step (b), which can disaggregate particle aggregates in the mixture. This method of making the formulation is particularly useful for formulations used to make medium dose containers containing a dosage of the formulation, such as about 3 mg to about 240 mg. The formulation can have, for example, from about 1% to about 70% egg white protein by weight.

[0055]

[0073] In some methods of making an egg white protein formulation, the method includes (a) mixing a dry egg white protein powder with a first diluent and a lubricant to form a first mixture; (b) mixing a second diluent and the first mixture at a higher shear force than that used in mixing in step (a) to form a second mixture; and (c) mixing the second mixture and the lubricant to form the egg white protein formulation. The mixing in step (b) is performed such that the mixing disaggregates particle aggregates. This method of making the formulation is particularly useful for formulations used to make high-dose dosage containers, such as those containing doses of about 200 mg or more (e.g., up to 600 mg, or up to 300 mg). The formulation can have, for example, about 50% to about 80% egg white protein by weight. The egg white protein formulations described herein comprise, consist essentially of, or consist of dry egg white protein powder, one or more diluents (e.g., two diluents), and a lubricant. In some embodiments, the egg white protein preparations are substantially free of lubricants or substantially free of colloidal silicon dioxide. Optionally, the egg white protein preparations comprise, consist essentially of, or consist of dry egg white protein powder, one or more diluents (e.g., two diluents), a lubricant, and a lubricant (such as colloidal silicon dioxide).

[0056]

[0074] The egg white protein formulation can be packaged in a dosage container, such as a capsule or sachet. The dose of egg white protein in the dosage container depends on the amount of formulation packaged in the container and the concentration of the egg white protein in the formulation. For example, the dose of egg white protein in the dosage container can be from 0.1 mg to about 1000 mg of egg white protein.

[0057]

[0075] The dried egg white protein powder used to manufacture the formulation, the manufactured egg white protein formulation, and the manufactured dosage container containing the egg white protein formulation should be carefully controlled to ensure accurate administration of egg white protein doses to patients being treated for egg allergy by oral immunotherapy. As further described herein, one or more methods can be used to evaluate the quality of the dried egg white protein powder, the egg white protein formulation, or the dosage container. Such methods include measuring the protein content of the powder or formulation, characterizing one or more allergenic egg white proteins (e.g., ovalbumin, ovomucoid, ovotransferrin, and / or lysozyme) in the powder or formulation, measuring the blend uniformity of the formulation, measuring the deliverable amount of the formulation from a lot of dosage containers, or measuring the content uniformity within a lot of dosage containers.

[0058]

[0076] Further described herein is a method of treating egg allergy in a patient, the method comprising orally administering to the patient multiple doses of a pharmaceutical composition comprising egg white protein according to an oral immunotherapy schedule, the oral immunotherapy schedule including: (a) a dose escalation period comprising orally administering to the patient a series of ascending doses of about 1 mg to about 300 mg of egg white protein, where a given dose is administered to the patient for at least 2 weeks before the dose is escalated, the dose escalation period being about 20 weeks to about 44 weeks in length; and (b) a maintenance period comprising orally administering to the patient multiple maintenance doses comprising egg white protein, where the maintenance period is about 12 weeks or more in length.

[0059]

[0077] The dosage of the pharmaceutical composition administered to the patient may be adjusted (e.g., by decreasing the dosage, skipping a dosage, delaying a dosage (in whole or in part), or delaying an increase in dosage) in response to an adverse event associated with administration of the pharmaceutical composition, or some other concurrent factor associated with increased sensitivity to an allergen unrelated to administration of the pharmaceutical composition to the patient (such as atopic disease flare-up, inflammation, disease stage, menstruation, or unintentional exposure to a food to which the patient is allergic).

[0060]

[0078] The oral immunotherapy (OIT) treatment schedule described herein is designed to safely desensitize a patient's allergy to egg proteins such that the patient can ingest egg proteins by accidental exposure without moderate or severe allergenic adverse events. Patients can be maintained on a cooked egg avoidance diet post-treatment depending on the individual patient's treatment outcome.

[0061] definition

[0079] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0062]

[0080] Reference herein to "about" a value or parameter includes (and describes) the variation that is directed to that value or parameter itself. For example, a description that refers to "about X" includes a description of "X."

[0063]

[0081] An "adverse event" is any undesirable and unintended sign (including abnormal laboratory findings), symptom, or disease, or aggravation thereof, temporarily associated with oral immunotherapy, whether or not related to oral immunotherapy.

[0064]

[0082] A composition "consisting essentially of" a recited material includes only at least 99.95% of that material by weight of the composition. Additives, impurities, or other components may comprise less than 0.05% by weight of the composition.

[0065]

[0083] The term "cooked" refers to heat treatment of the composition without baking the composition, resulting in at least partial modification or denaturation of one or more proteins in the composition. The term "baked" refers to heat treatment of the composition at a temperature of at least 176°C for at least 20 minutes without baking the composition.

[0066]

[0084] The term "desensitized" is used herein to refer to an increase in the allergic reaction threshold to food allergens by a subject as a result of oral immunotherapy against food allergens.Desensitization to food allergens can be tested using methods known in the art, including oral food challenge.Desensitization may be partial, where the subject tolerates increased amounts of food allergens compared to before treatment, but still responds to high doses of food allergens, or desensitization may be complete, where the patient tolerates all tested doses of food allergens.

[0067]

[0085] The terms "effective," "efficacy," or "effectiveness," unless otherwise indicated, are used herein to refer to the ability of a treatment to induce immune modulation, such as desensitization, or to maintain a desired immune state, such as a desensitized state.

[0068]

[0086] An "escalating dose" is the first dose administered to a patient during the course of oral immunotherapy that is higher than the immediately preceding dose administered to the patient.

[0069]

[0087] As used herein, "maintenance period" refers to the phase of egg protein oral immunotherapy that involves administration of egg white protein (i.e., a maintenance dose) to the patient and occurs after completion of the dose escalation period.

[0070]

[0088] As used herein, "mild allergic adverse event" refers to an observed or experienced OIT treatment-related allergic adverse event that is associated with temporary discomfort, but does not require immediate medical intervention such as hospitalization or epinephrine, and does not substantially interfere with daily activities.

[0071]

[0089] As used herein, a "moderate allergic adverse event" refers to an observed or experienced OIT treatment-related allergic adverse event that is associated with sufficient discomfort to interfere with daily activities and may prompt medical intervention and / or further observation.

[0072]

[0090] As used herein, the phrase "severe allergic adverse event" refers to an observed or experienced OIT treatment-related allergic adverse event that required hospitalization and / or administration of epinephrine or other life-saving medical intervention.

[0073]

[0091] The terms "subject" and "patient" are used interchangeably herein to describe a human of any age.

[0074]

[0092] A composition is "substantially free" of a material if the composition contains less than 0.005% by weight of that material or is free of that material.

[0075]

[0093] A patient "tolerates" a dose when the dose is administered to the patient and completely ingested by the patient without any moderate or severe allergic adverse events in response to the dose. A patient is considered to tolerate a dose even if a mild allergic adverse event is observed or experienced. A "maximum tolerated dose" is the maximum dose administered to a patient during an oral food challenge that is tolerated by the patient without any moderate or severe allergic adverse events. A "cumulative tolerated dose" is the sum of doses administered to a patient during an oral food challenge up to and including the maximum tolerated dose without any moderate or severe allergic adverse events.

[0076]

[0094] The terms "treat," "treating," and "treatment" are used interchangeably herein to refer to any action that provides a benefit to a subject afflicted with a disease state or condition, including amelioration of the condition through reduction, inhibition, suppression, or elimination of at least one symptom, slowing the progression of the disease, slowing the recurrence of the disease, inhibiting the disease, or partially or completely reducing the response or reaction to an allergen.

[0077]

[0095] "Dose escalation period" refers to a phase of oral immunotherapy characterized by a series of increasing food allergen doses that begins with administration of a dose of food allergen lower than the maximum dose administered to a patient during oral immunotherapy and ends when the maximum dose administered to a patient during oral immunotherapy is achieved.

[0078]

[0096] The terms "weight percentage," "weight percent," and "wt. %" are used interchangeably herein and refer to the proportion of a recited component of a composition relative to the total weight of the composition.

[0079]

[0097] It will be understood that the aspects and variations of the invention described herein include those that "consist" and / or "consist essentially of" aspects and variations.

[0080]

[0098] When a range of values ​​is provided, it is understood that each intervening value between the upper and lower limit of that range, and any other stated or intervening value within that stated range, is encompassed within the scope of the disclosure. When a stated range includes an upper or lower limit, ranges excluding any of those included limits are also included in the disclosure.

[0081]

[0099] It should be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present invention. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0082]

[0100] The features and preferences described above in connection with the "embodiments" are of different preferences and are not limited to that particular embodiment only. They may be freely combined with features from other embodiments where technically feasible to form preferred combinations of features. This specification is presented to enable any person skilled in the art to make and use the invention and is provided in the context of a patent application and its requirements. Various modifications to the described embodiments will be readily apparent to those skilled in the art, and the generic principles of the specification may be applied to other embodiments. Thus, the present invention is not intended to be limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features described herein.

[0083]

[0101] The disclosures of all publications, patents, and patent applications referenced herein are each incorporated herein by reference in their entirety. To the extent that any reference incorporated by reference conflicts with the present disclosure, the present disclosure shall control.

[0084] Egg white protein preparations

[0102] The egg white protein formulations described herein include dried egg white (DEW) protein powder and can be formulated with one or more excipients. Typically, the formulations include one or more different diluents (preferably, two different diluents) and a lubricant. Some egg white protein formulations further include a lubricant, but the lubricant may be omitted in certain formulations. For example, the lubricant is generally omitted in low-dose formulations (e.g., formulations having less than about 2.5% by weight of egg white protein). Thus, in some formulations, the egg white protein formulation is substantially free of lubricant or substantially free of colloidal silicon dioxide. The excipients and the ratio of excipients may be selected according to the dose of egg white protein when packaged in a dosage container.

[0085]

[0103] Dried egg white protein powder is typically derived from poultry (preferably chicken (Gallus domesticus)) egg whites. Dried egg white protein powder is frequently used as a food ingredient, but can be formulated for use as an oral immunotherapy formulation to treat egg allergies. The use of non-formulated dried egg white protein powder for oral immunotherapy treatment is not encouraged, because safe treatment requires careful dosing, especially at low doses, which are difficult to obtain consistently without formulation. Dried egg white protein powder generally has a protein content of about 80% to about 85%, but the amount of dried egg white protein powder added to a formulation lot can be adjusted based on the actual protein content of the dried egg white protein powder, with concomitant adjustments to the amount of one or more excipients (such as one of the diluents, such as pregelatinized starch) to obtain the desired dosage level. For example, the protein content of the dried egg white protein powder can be about 50% to about 90% by weight (such as about 70% to about 90% by weight, about 75% to about 90% by weight, or about 70% to about 85% by weight). The remaining weight of the dried egg white protein powder may include small amounts of fat, carbohydrates, vitamins, minerals, or other components naturally occurring in egg whites. The dried egg white protein powder may further include residual water.

[0086]

[0104] Dried egg white protein powder is derived from liquid egg white, generally by spray drying dried egg white. Preferably, the dried egg white protein powder is pasteurized or sterilized (e.g., by irradiation), but care should be taken to ensure that the allergenic proteins are not denatured during the manufacturing process. Although spray drying and pasteurization are not expected to denature the proteins, quality control processes (such as measuring the potency or relative potency of one or more allergenic proteins, such as ovalbumin, ovotransferrin, ovomucoid, and / or lysozyme) can be used to evaluate the dried egg white protein powder and / or the manufactured egg white protein preparation. Polyacrylamide gel electrophoresis (PAGE), such as SDS-PAGE, or immunoblots can also be used to detect the degradation of lysozyme or ovotransferrin, which disappears or becomes weaker from the gel when degraded.

[0087]

[0105] The dried egg white protein powder can be produced by separating the liquid egg white and the egg yolk from a cracked egg. The pH of the egg white can be adjusted as needed using a suitable acid or base known in the art. Glucose can be removed from the liquid egg white, which can reduce browning of the dried egg white protein powder that can occur via the Maillard reaction. Glucose can be removed, for example, by adding glucose oxidase, catalase, and hydrogen peroxide solution to the liquid egg white, or by fermentation (e.g., by adding yeast to the egg white).

[0088]

[0106] To dry the liquid egg white, it can be passed through a spray dryer. The liquid egg white is sprayed through a nozzle of the spray dryer into a chamber and dried using a heated air stream. The amount of heat and length of exposure is set to dry the liquid egg white into a powder but not to denature the allergenic proteins in the egg white (e.g., lysozyme, ovalbumin, ovomucoid, and ovotransferrin).

[0089]

[0107] The dried egg white protein powder can be pasteurized after the drying process. Pasteurization kills harmful bacteria such as Salmonella, but does not denature the allergenic proteins in the dried egg white protein powder. To pasteurize the dried egg white protein powder, the powder can be held at about 50°C to about 60°C (preferably, about 54°C to about 60°C) for about 7 days or more. The pasteurization process continues at least until the dried egg white powder is negative for live Salmonella spp. and / or E. coli (either of which can be assessed, for example, by separate culture or other suitable means).

[0090]

[0108] Dried egg white protein powder may include materials that are added during the process of processing the powder, such as glucose oxidase, catalase, hydrogen peroxide, acids or bases used to adjust the pH, or vehicles for any of the components (e.g., salts, buffers, stabilizers, etc.). However, these additives are relatively small, and it is generally preferred that the dried egg white protein powder be substantially free of additives prior to production of the egg white protein formulation, in which the formulation excipients are mixed with the dried egg white protein powder.

[0091]

[0109] Because egg white protein formulations are manufactured by combining and blending powders, particle size of the dried egg white protein powder was identified as a potential factor that can affect the blend uniformity of the batch produced formulation and / or the content uniformity of the batch in the dosage container, especially at the lowest dosage strengths. Powder particle size can affect the blending process, adhesion of particles to carrier excipients or processing equipment surfaces, or segregation after the blending process. The median particle size (d 50 ) is generally about 30 μm to about 150 μm (about 30 μm to about 50 μm, about 50 μm to about 80 μm, about 80 μm to about 120 μm, or about 120 μm to about 150 μm, etc.). 10 is typically about 3 μm or more (and less than the median particle size). 90is typically about 250 μm or less (and greater than the median particle size), such as about 200 μm or less, or about 175 μm or less. 50 , d 90 , d 10 ) refers to the particle size at a reference percentile (i.e., 50th, 90th, or 10th) by accumulated mass (or accumulated volume equivalent to the mass of a uniformly dense particle) or accumulated number. Particle size can be measured using standard laser diffraction techniques (e.g., dynamic laser scattering), where the particles are suspended in a solvent that does not dissolve the particles (such as methanol, ethanol, or other suitable solvent).

[0092]

[0110] The excipients included in the egg white protein formulation are selected so that the formulation can be packaged (e.g., in capsules, sachets, or other suitable packaging) with an accurate and consistent content of egg white protein. The excipients of the formulation include one or more (preferably two) different diluents, and a lubricant. In some formulations, a lubricant is included. However, in some embodiments, a lubricant is omitted from the formulation. For example, in some formulations (such as low-dose formulations having less than about 5% by weight of egg white protein), the egg white protein formulation is free or substantially free of colloidal silicon dioxide. Representative diluents included in the formulation include pregelatinized starch and microcrystalline cellulose, or a combination thereof. Magnesium stearate has been found to be a suitable lubricant and is commonly included in the formulation. The lubricant, if included in the formulation, is preferably colloidal silicon dioxide.

[0093]

[0111] The egg white protein formulation is packaged in a dosage container, and the amount of the egg white protein formulation and the concentration of the egg white protein in the formulation determine the dose of the egg white protein in the dosage container. The dosage container is not ingested in its entirety, but instead the contents are removed prior to ingestion. Once removed, the egg white protein formulation can be mixed with a food vehicle (such as yogurt, oatmeal, pudding, applesauce, or other suitable food vehicle) to aid in ingestion. As dose accuracy is particularly important when administering oral immunotherapy, the egg white protein formulation should have good flow properties to ensure that an appropriate portion of the contents of the container is removed and ingested. The formulations described herein allow the egg white protein formulation in the container to properly flow out of the container prior to ingestion.

[0094]

[0112] Pregelatinized starch and microcrystalline cellulose are particularly useful diluents because they are free-flowing powders that mix with egg white protein powder. A higher percentage of microcrystalline cellulose is included in lower egg white protein dose formulations (e.g., doses of about 10 mg or less) to ensure good flowability and bulk density of the egg white protein formulation for packaging in and removal from containers, such as packaging in and removal from capsules. For example, a low-dose (e.g., about 10 mg or less) egg white protein preparation may contain about 25% by weight to about 60% by weight (e.g., about 25% by weight to about 30% by weight, about 30% by weight to about 40% by weight, about 40% by weight to about 50% by weight, or about 50% by weight to about 60% by weight, etc.) of microcrystalline cellulose, while a high-dose (e.g., greater than about 10 mg) egg white protein preparation may contain about 5% by weight to about 25% by weight (e.g., about 5% by weight to about 10% by weight, about 10% by weight to about 15% by weight, about 15% by weight to about 20% by weight, or about 20% by weight to about 25% by weight, etc.) of microcrystalline cellulose.

[0095]

[0113] Magnesium stearate has been found to be a useful lubricant to prevent adhesion of egg white protein to equipment surfaces during the manufacturing process. In general, egg white protein formulations contain about 0.2% to about 2% by weight of magnesium stearate, e.g., about 0.2% to about 0.4% by weight, about 0.4% to about 0.6% by weight, about 0.6% to about 1% by weight, about 1% to about 1.5% by weight, or about 1.5% to about 2% by weight. In some embodiments, the egg white protein formulations contain about 0.5% by weight of magnesium stearate.

[0096]

[0114] A lubricant such as colloidal silicon dioxide may be included in high dose egg white protein formulations (e.g., a dose of egg white protein greater than about 6 mg or a dose of egg white protein greater than about 12 mg). Omission of the lubricant from larger doses was found to result in a portion of the egg white protein formulation remaining in the container, which results in underdosing of the patient. However, including the lubricant in the egg white formulation allowed substantially all of the egg white protein formulation to be delivered from the container when poured. The addition of colloidal silicon dioxide was found to form soft low density aggregates. A high shear mixing process (such as a conical mill) was one solution found to improve the content uniformity of formulations containing colloidal silicon dioxide. Preferably, the high shear mixing process that breaks down these low density aggregates does not change the primary particle size of any blend components. Thus, a lubricant such as colloidal silicon dioxide may be included in the egg white protein formulation in an amount of, for example, about 0.2 wt% to about 2 wt% (such as about 0.2 wt% to about 0.4 wt%, about 0.4 wt% to about 0.6 wt%, about 0.6 wt% to about 1 wt%, about 1 wt% to about 1.5 wt%, or about 1.5 wt% to about 2 wt%). By including a lubricant, the deliverability of the egg white protein powder from the container can be increased at higher doses. In some embodiments, about 95 wt% or more (such as about 96 wt% or more, about 97 wt% or more, about 98 wt% or more, about 99 wt% or more, or about 99.5 wt% or more) of the egg white protein formulation in the container is deliverable from the container. Furthermore, the egg white protein formulation can be reliably delivered from the container. For example, in some embodiments, the dosage container is selected from a batch comprising multiple dosage containers, and the average deliverable egg white protein formulation is about 95% by weight or more (such as about 96% by weight or more, about 97% by weight or more, about 98% by weight or more, about 99% by weight or more, or about 99.5% by weight or more).

[0097]

[0115] For low-dose (e.g., about 6 mg or less of egg white protein) egg white protein formulations, silicon dioxide has been found to interfere with quality control analysis, particularly quantification of protein concentration. As previously mentioned, the amount of egg white protein contained in a dosage container should be precise to minimize the risk of accidental overdosing, especially when low doses are administered to a subject. Furthermore, it has been further discovered that a lubricant included in a high-dose egg white protein formulation to ensure sufficient deliverability from a dosage container can be omitted in an egg white protein formulation used at a low dose, while maintaining sufficient deliverability from the container. In some embodiments, about 95% by weight or more (e.g., about 96% by weight or more, about 97% by weight or more, about 98% by weight or more, about 99% by weight or more, or about 99.5% by weight or more) of the egg white protein formulation in a dosage container containing 6 mg or less of egg white protein (e.g., about 0.1 mg to about 6 mg of egg white protein) is deliverable from the container, even when the egg white protein formulation does not contain colloidal silicon dioxide. Furthermore, the egg white protein formulation can be reliably delivered from the container. For example, in some embodiments, the dosage container is selected from a batch comprising multiple dosage containers, and the average deliverable egg white protein formulation is about 95% by weight or more (such as about 96% by weight or more, about 97% by weight or more, about 98% by weight or more, about 99% by weight or more, or about 99.5% by weight or more).

[0098] Dosage of egg white protein preparations

[0116] The egg white protein preparations described herein are packaged in dosage containers such as capsules or sachets. During the course of oral immunotherapy, different doses of egg white protein are administered to patients with egg allergy, which are selected based on the treatment phase and / or tolerability of the egg white protein. Briefly, the patient orally ingests increasing amounts of egg white protein during a dose escalation period (usually by daily administration of the egg white protein preparation, with periodic dose escalation (e.g., dose increases every two weeks)), which is followed by a maintenance period at a higher dose level. Further description of exemplary oral immunotherapy administration schedules is provided herein. For administration, the egg white protein preparation is removed from the dosage container and orally ingested. For example, the entire capsule containing the egg white protein preparation is not ingested, but instead, the egg white protein preparation is removed from the capsule prior to oral administration to the patient. Preferably, the egg white protein preparation is mixed with a food vehicle prior to ingestion.

[0099]

[0117] The amount of an egg white protein formulation having a given egg white protein concentration packaged in a dosage container sets the amount of egg white protein (i.e., dose) in that dosage container. Thus, the dose of the container is a function of the egg white protein concentration in the formulation and the amount of formulation added to the container. The intended dose (i.e., label claim) of egg white protein in a dosage container can range, for example, from about 0.1 mg to about 600 mg of egg white protein, or any amount within this range. By way of example, the dose (label claim) of a dosage container can be 0.2 mg, 1 mg, 3 mg, 6 mg, 12 mg, 20 mg, 40 mg, 80 mg, 120 mg, 160 mg, 200 mg, 240 mg, or 300 mg of egg white protein.

[0100]

[0118] The amount of the egg white protein preparation in the container is limited by the size of the container, but is generally in the range of about 100 mg to about 1000 mg, for example, about 100 mg to about 250 mg, about 250 mg to about 400 mg, about 400 mg to about 600 mg, or about 600 mg to about 1000 mg. For example, in some embodiments, about 180 mg of the egg white protein preparation is contained in the container (such as a capsule). In some embodiments, about 500 mg of the egg white protein preparation is contained in the container (such as a capsule or sachet). Smaller containers may be more suitable for smaller dose sizes. For example, an egg white protein preparation for obtaining a dose of about 0.1 mg to about 10 mg (or about 0.2 mg to about 6 mg) of egg white protein can be contained in a container having a capacity of about 180 mg (e.g., two capsule size). Larger doses of the egg white protein formulation can be contained in larger container sizes, for example, doses of about 12 mg to about 300 mg can be contained in a container having a capacity of about 500 mg (e.g., a 00 capsule size). Exemplary capsule sizes can be 000, 00, 0, 1, 2, or 3.

[0101]

[0119] Since the dosage container itself is not ingested (as the contents of the container are ingested), the material of the container does not need to be edible. However, in case of inadvertent ingestion of the container, it may be useful to have an edible container. The container preferably limits the exposure of the contents to moisture or air. An exemplary container may be a hypromellose-based container (such as a capsule) or a foil-lined sachet. The container must be easily openable so that the egg white protein formulation in the container can flow out of the container.

[0102]

[0120] In one example, the egg white protein formulation comprises, consists essentially of, or consists of about 0.1% to about 0.3% by weight of dry egg white protein powder, about 50% to about 70% by weight of a first diluent (such as pregelatinized starch), about 35% to about 45% by weight of a second diluent (such as microcrystalline cellulose), and about 0.25% to about 0.75% by weight of a lubricant (such as magnesium stearate). In some embodiments, the egg white protein formulation is substantially free of lubricants or substantially free of colloidal silicon dioxide. In some embodiments, the egg white protein formulation comprises about 0.08% to about 0.24% by weight of egg white protein. The egg white protein formulation may be packaged in a dosage container (such as a capsule, e.g., a size 2 capsule) having an intended egg white protein dose (i.e., label claim) of 0.2 mg. The dosage container may contain about 170 mg to about 190 mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage container may be from about 0.15 mg to about 0.25 mg (e.g., from about 0.17 mg to about 0.23 mg, from about 0.18 mg to about 0.22 mg, or from about 0.19 mg to about 0.21 mg).

[0103]

[0121] In another example, the egg white protein formulation comprises, consists essentially of, or consists of about 0.1% to about 0.3% by weight of dry egg white protein powder, about 50% to about 70% by weight of pregelatinized starch, about 35% to about 45% by weight of microcrystalline cellulose, and about 0.25% to about 0.75% by weight of magnesium stearate. In some embodiments, the egg white protein formulation is substantially free of lubricants or substantially free of colloidal silicon dioxide. In some embodiments, the egg white protein formulation comprises about 0.08% to about 0.24% by weight of egg white protein. The egg white protein formulation may be packaged in a dosage container (such as a capsule, e.g., a size 2 capsule) having an intended egg white protein dose (i.e., label claim) of 0.2 mg. The dosage container may contain about 170 mg to about 190 mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage container may be from about 0.15 mg to about 0.25 mg (e.g., from about 0.17 mg to about 0.23 mg, from about 0.18 mg to about 0.22 mg, or from about 0.19 mg to about 0.21 mg).

[0104]

[0122] In another example, the egg white protein formulation comprises, consists essentially of, or consists of about 0.5% to about 0.8% by weight of dry egg white protein powder, about 50% to about 70% by weight of a first diluent (such as pregelatinized starch), about 35% to about 45% by weight of a second diluent (such as microcrystalline cellulose), and about 0.25% to about 0.75% by weight of a lubricant (such as magnesium stearate). In some embodiments, the egg white protein formulation is substantially free of lubricants or substantially free of colloidal silicon dioxide. In some embodiments, the egg white protein formulation comprises about 0.4% to about 0.64% by weight of egg white protein. The egg white protein formulation may be packaged in a dosage container (such as a capsule, e.g., a size 2 capsule) having an intended egg white protein dose (i.e., label claim) of 1 mg. The dosage container may contain about 170 mg to about 190 mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage container may be from about 0.75 mg to about 1.25 mg (e.g., from about 0.85 mg to about 1.15 mg, from about 0.9 mg to about 1.1 mg, or from about 0.95 mg to about 1.05 mg).

[0105]

[0123] In another example, the egg white protein formulation comprises, consists essentially of, or consists of about 0.5% to about 0.8% by weight of dry egg white protein powder, about 50% to about 70% by weight of pregelatinized starch, about 35% to about 45% by weight of microcrystalline cellulose, and about 0.25% to about 0.75% by weight of magnesium stearate. In some embodiments, the egg white protein formulation is substantially free of lubricants or substantially free of colloidal silicon dioxide. In some embodiments, the egg white protein formulation comprises about 0.4% to about 0.64% by weight of egg white protein. The egg white protein formulation may be packaged in a dosage container (such as a capsule, e.g., a size 2 capsule) having an intended egg white protein dose (i.e., label claim) of 1 mg. The dosage container may contain about 170 mg to about 190 mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage container may be from about 0.75 mg to about 1.25 mg (e.g., from about 0.85 mg to about 1.15 mg, from about 0.9 mg to about 1.1 mg, or from about 0.95 mg to about 1.05 mg).

[0106]

[0124] In another example, the egg white protein formulation comprises, consists essentially of, or consists of about 1.8% to about 2.4% by weight of dry egg white protein powder, about 50% to about 70% by weight of a first diluent (such as pregelatinized starch), about 35% to about 45% by weight of a second diluent (such as microcrystalline cellulose), and about 0.25% to about 0.75% by weight of a lubricant (such as magnesium stearate). In some embodiments, the egg white protein formulation is substantially free of lubricants or substantially free of colloidal silicon dioxide. In some embodiments, the egg white protein formulation comprises about 1.44% to about 1.92% by weight of egg white protein. The egg white protein formulation may be packaged in a dosage container (such as a capsule, e.g., a size 2 capsule) having an intended egg white protein dose (i.e., label claim) of 3 mg. The dosage container may contain about 170 mg to about 190 mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage container may be from about 2.25 mg to about 3.75 mg (e.g., from about 2.55 mg to about 3.45 mg, from about 2.7 mg to about 3.3 mg, or from about 2.85 mg to about 3.15 mg).

[0107]

[0125] In another example, the egg white protein formulation comprises, consists essentially of, or consists of about 1.8% to about 2.4% by weight of dry egg white protein powder, about 50% to about 70% by weight of pregelatinized starch, about 35% to about 45% by weight of microcrystalline cellulose, and about 0.25% to about 0.75% by weight of magnesium stearate. In some embodiments, the egg white protein formulation is substantially free of lubricants or substantially free of colloidal silicon dioxide. In some embodiments, the egg white protein formulation comprises about 1.44% to about 1.92% by weight of egg white protein. The egg white protein formulation may be packaged in a dosage container (such as a capsule, e.g., a size 2 capsule) having an intended egg white protein dose (i.e., label claim) of 3 mg. The dosage container may contain about 170 mg to about 190 mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage container may be from about 2.25 mg to about 3.75 mg (e.g., from about 2.55 mg to about 3.45 mg, from about 2.7 mg to about 3.3 mg, or from about 2.85 mg to about 3.15 mg).

[0108]

[0126] In another example, the egg white protein formulation comprises, consists essentially of, or consists of about 4% to about 4.5% by weight of dry egg white protein powder, about 45% to about 65% by weight of a first diluent (such as pregelatinized starch), about 35% to about 45% by weight of a second diluent (such as microcrystalline cellulose), and about 0.25% to about 0.75% by weight of a lubricant (such as magnesium stearate). In some embodiments, the egg white protein formulation is substantially free of lubricants or substantially free of colloidal silicon dioxide. In some embodiments, the egg white protein formulation comprises about 1.44% to about 1.92% by weight of egg white protein. The egg white protein formulation may be packaged in a dosage container (such as a capsule, e.g., a size 2 capsule) having an intended egg white protein dose (i.e., label claim) of 6 mg. The dosage container may contain about 170 mg to about 190 mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage container may be from about 4.5 mg to about 7.5 mg (e.g., from about 5.1 mg to about 6.9 mg, from about 5.4 mg to about 6.6 mg, from about 5.7 mg to about 6.3 mg, or from about 5.8 mg to about 6.2 mg).

[0109]

[0127] In another example, the egg white protein formulation comprises, consists essentially of, or consists of about 4% to about 4.5% by weight of dry egg white protein powder, about 45% to about 65% by weight of pregelatinized starch, about 35% to about 45% by weight of microcrystalline cellulose, and about 0.25% to about 0.75% by weight of magnesium stearate. In some embodiments, the egg white protein formulation is substantially free of lubricants or substantially free of colloidal silicon dioxide. In some embodiments, the egg white protein formulation comprises about 1.44% to about 1.92% by weight of egg white protein. The egg white protein formulation may be packaged in a dosage container (such as a capsule, e.g., a size 2 capsule) having an intended egg white protein dose (i.e., label claim) of 6 mg. The dosage container may contain about 170 mg to about 190 mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage container may be from about 4.5 mg to about 7.5 mg (e.g., from about 5.1 mg to about 6.9 mg, from about 5.4 mg to about 6.6 mg, from about 5.7 mg to about 6.3 mg, or from about 5.8 mg to about 6.2 mg).

[0110]

[0128] In another example, the egg white protein formulation comprises, consists essentially of, or consists of about 2.5% to about 3.5% by weight of dry egg white protein powder, about 70% to about 85% by weight of a first diluent (such as pregelatinized starch), about 10% to about 20% by weight of a second diluent (such as microcrystalline cellulose), about 0.25% to about 0.75% by weight of a lubricant (such as magnesium stearate), and about 0.25% to about 0.75% by weight of a lubricant (such as colloidal silicon dioxide). In some embodiments, the egg white protein formulation comprises about 2% to about 2.8% by weight of egg white protein. The egg white protein formulation may be packaged in a dosage container (such as a capsule, e.g., a size 00 capsule) having an intended egg white protein dose (i.e., label claim) of 12 mg. The dosage container may contain about 480 mg to about 520 mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage container may be from about 9 mg to about 15 mg (e.g., from about 10.2 mg to about 13.8 mg, from about 10.8 mg to about 13.2 mg, from about 11.4 mg to about 12.6 mg, or from about 11.7 mg to about 12.3 mg).

[0111]

[0129] In another example, the egg white protein formulation comprises, consists essentially of, or consists of about 2.5% to about 3.5% by weight of dry egg white protein powder, about 70% to about 85% by weight of pregelatinized starch, about 10% to about 20% by weight of microcrystalline cellulose, about 0.25% to about 0.75% by weight of magnesium stearate, and about 0.25% to about 0.75% by weight of colloidal silicon oxide. In some embodiments, the egg white protein formulation comprises about 2% to about 2.8% by weight of egg white protein. The egg white protein formulation may be packaged in a dosage container (such as a capsule, e.g., a size 00 capsule) having an intended egg white protein dose (i.e., label claim) of 12 mg. The dosage container may contain about 480 mg to about 520 mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage container may be from about 9 mg to about 15 mg (e.g., from about 10.2 mg to about 13.8 mg, from about 10.8 mg to about 13.2 mg, from about 11.4 mg to about 12.6 mg, or from about 11.7 mg to about 12.3 mg).

[0112]

[0130] In another example, the egg white protein formulation comprises, consists essentially of, or consists of about 4.5% to about 5.5% by weight of dry egg white protein powder, about 70% to about 85% by weight of a first diluent (such as pregelatinized starch), about 10% to about 20% by weight of a second diluent (such as microcrystalline cellulose), about 0.25% to about 0.75% by weight of a lubricant (such as magnesium stearate), and about 0.25% to about 0.75% by weight of a lubricant (such as colloidal silicon dioxide). In some embodiments, the egg white protein formulation comprises about 3.6% to about 4.4% by weight of egg white protein. The egg white protein formulation may be packaged in a dosage container (such as a capsule, e.g., a size 00 capsule) having an intended egg white protein dose (i.e., label claim) of 20 mg. The dosage container may contain about 480 mg to about 520 mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage container may be from about 15 mg to about 25 mg (e.g., from about 17 mg to about 23 mg, from about 18 mg to about 22 mg, from about 19 mg to about 21 mg, or from about 19.5 mg to about 20.5 mg).

[0113]

[0131] In another example, the egg white protein formulation comprises, consists essentially of, or consists of about 4.5% to about 5.5% by weight of dry egg white protein powder, about 70% to about 85% by weight of pregelatinized starch, about 10% to about 20% by weight of microcrystalline cellulose, about 0.25% to about 0.75% by weight of magnesium stearate, and about 0.25% to about 0.75% by weight of colloidal silicon dioxide. In some embodiments, the egg white protein formulation comprises about 3.6% to about 4.4% by weight of egg white protein. The egg white protein formulation may be packaged in a dosage container (such as a capsule, e.g., a size 00 capsule) having an intended egg white protein dose (i.e., label claim) of 20 mg. The dosage container may contain about 480 mg to about 520 mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage container may be from about 15 mg to about 25 mg (e.g., from about 17 mg to about 23 mg, from about 18 mg to about 22 mg, from about 19 mg to about 21 mg, or from about 19.5 mg to about 20.5 mg).

[0114]

[0132] In another example, the egg white protein formulation comprises, consists essentially of, or consists of about 8% to about 12% by weight of dry egg white protein powder, about 65% to about 85% by weight of a first diluent (such as pregelatinized starch), about 10% to about 20% by weight of a second diluent (such as microcrystalline cellulose), about 0.25% to about 0.75% by weight of a lubricant (such as magnesium stearate), and about 0.25% to about 0.75% by weight of a lubricant (such as colloidal silicon dioxide). In some embodiments, the egg white protein formulation comprises about 6.4% to about 9.6% by weight of egg white protein. The egg white protein formulation may be packaged in a dosage container (such as a capsule, e.g., a size 00 capsule) having an intended egg white protein dose (i.e., label claim) of 40 mg. The dosage container may contain about 480 mg to about 520 mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage container may be from about 30 mg to about 50 mg (e.g., from about 34 mg to about 46 mg, from about 36 mg to about 44 mg, from about 38 mg to about 42 mg, or from about 39 mg to about 41 mg).

[0115]

[0133] In another example, the egg white protein formulation comprises, consists essentially of, or consists of about 8% to about 12% by weight of dry egg white protein powder, about 65% to about 85% by weight of pregelatinized starch, about 10% to about 20% by weight of microcrystalline cellulose, about 0.25% to about 0.75% by weight of magnesium stearate, and about 0.25% to about 0.75% by weight of colloidal silicon dioxide. In some embodiments, the egg white protein formulation comprises about 6.4% to about 9.6% by weight of egg white protein. The egg white protein formulation may be packaged in a dosage container (such as a capsule, e.g., a size 00 capsule) having an intended egg white protein dose (i.e., label claim) of 40 mg. The dosage container may contain about 480 mg to about 520 mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage container may be from about 30 mg to about 50 mg (e.g., from about 34 mg to about 46 mg, from about 36 mg to about 44 mg, from about 38 mg to about 42 mg, or from about 39 mg to about 41 mg).

[0116]

[0134] In another example, the egg white protein formulation comprises, consists essentially of, or consists of about 16% to about 24% by weight of dry egg white protein powder, about 55% to about 75% by weight of a first diluent (such as pregelatinized starch), about 10% to about 20% by weight of a second diluent (such as microcrystalline cellulose), about 0.25% to about 0.75% by weight of a lubricant (such as magnesium stearate), and about 0.25% to about 0.75% by weight of a lubricant (such as colloidal silicon dioxide). In some embodiments, the egg white protein formulation comprises about 12.8% to about 19.2% by weight of egg white protein. The egg white protein formulation may be packaged in a dosage container (such as a capsule, e.g., a size 00 capsule) having an intended egg white protein dose (i.e., label claim) of 80 mg. The dosage container may contain about 480 mg to about 520 mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage container may be from about 60 mg to about 100 mg (e.g., from about 68 mg to about 92 mg, from about 72 mg to about 88 mg, from about 76 mg to about 84 mg, or from about 78 mg to about 82 mg).

[0117]

[0135] In another example, the egg white protein formulation comprises, consists essentially of, or consists of about 16% to about 24% by weight of dry egg white protein powder, about 55% to about 75% by weight of pregelatinized starch, about 10% to about 20% by weight of microcrystalline cellulose, about 0.25% to about 0.75% by weight of magnesium stearate, and about 0.25% to about 0.75% by weight of colloidal silicon dioxide. In some embodiments, the egg white protein formulation comprises about 12.8% to about 19.2% by weight of egg white protein. The egg white protein formulation may be packaged in a dosage container (such as a capsule, e.g., a size 00 capsule) having an intended egg white protein dose (i.e., label claim) of 80 mg. The dosage container may contain about 480 mg to about 520 mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage container may be from about 60 mg to about 100 mg (e.g., from about 68 mg to about 92 mg, from about 72 mg to about 88 mg, from about 76 mg to about 84 mg, or from about 78 mg to about 82 mg).

[0118]

[0136] In another example, the egg white protein formulation comprises, consists essentially of, or consists of about 24% to about 36% by weight of dry egg white protein powder, about 45% to about 65% by weight of a first diluent (such as pregelatinized starch), about 10% to about 20% by weight of a second diluent (such as microcrystalline cellulose), about 0.25% to about 0.75% by weight of a lubricant (such as magnesium stearate), and about 0.25% to about 0.75% by weight of a lubricant (such as colloidal silicon dioxide). In some embodiments, the egg white protein formulation comprises about 19.2% to about 28.8% by weight of egg white protein. The egg white protein formulation may be packaged in a dosage container (such as a capsule, e.g., a size 00 capsule) having an intended egg white protein dose (i.e., label claim) of 120 mg. The dosage container may contain about 480 mg to about 520 mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage container may be from about 90 mg to about 150 mg (e.g., from about 102 mg to about 138 mg, from about 108 mg to about 132 mg, from about 114 mg to about 126 mg, or from about 117 mg to about 123 mg).

[0119]

[0137] In another example, the egg white protein formulation comprises, consists essentially of, or consists of about 24% to about 36% by weight of dry egg white protein powder, about 45% to about 65% by weight of pregelatinized starch, about 10% to about 20% by weight of microcrystalline cellulose, about 0.25% to about 0.75% by weight of magnesium stearate, and about 0.25% to about 0.75% by weight of colloidal silicon dioxide. In some embodiments, the egg white protein formulation comprises about 19.2% to about 28.8% by weight of egg white protein. The egg white protein formulation may be packaged in a dosage container (such as a capsule, e.g., a size 00 capsule) having an intended egg white protein dose (i.e., label claim) of 120 mg. The dosage container may contain about 480 mg to about 520 mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage container may be from about 90 mg to about 150 mg (e.g., from about 102 mg to about 138 mg, from about 108 mg to about 132 mg, from about 114 mg to about 126 mg, or from about 117 mg to about 123 mg).

[0120]

[0138] In another example, the egg white protein formulation comprises, consists essentially of, or consists of about 32% to about 38% by weight of dry egg white protein powder, about 35% to about 55% by weight of a first diluent (such as pregelatinized starch), about 10% to about 20% by weight of a second diluent (such as microcrystalline cellulose), about 0.25% to about 0.75% by weight of a lubricant (such as magnesium stearate), and about 0.25% to about 0.75% by weight of a lubricant (such as colloidal silicon dioxide). In some embodiments, the egg white protein formulation comprises about 25.6% to about 30.4% by weight of egg white protein. The egg white protein formulation may be packaged in a dosage container (such as a capsule, e.g., a size 00 capsule) having an intended egg white protein dose (i.e., label claim) of 160 mg. The dosage container may contain about 480 mg to about 520 mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage container may be from about 120 mg to about 200 mg (e.g., from about 136 mg to about 184 mg, from about 144 mg to about 176 mg, from about 152 mg to about 168 mg, or from about 156 mg to about 154 mg).

[0121]

[0139] In another example, the egg white protein formulation comprises, consists essentially of, or consists of about 32% to about 38% by weight of dry egg white protein powder, about 35% to about 55% by weight of pregelatinized starch, about 10% to about 20% by weight of microcrystalline cellulose, about 0.25% to about 0.75% by weight of magnesium stearate, and about 0.25% to about 0.75% by weight of colloidal silicon dioxide. In some embodiments, the egg white protein formulation comprises about 25.6% to about 30.4% by weight of egg white protein. The egg white protein formulation may be packaged in a dosage container (such as a capsule, e.g., a size 00 capsule) having an intended egg white protein dose (i.e., label claim) of 160 mg. The dosage container may contain about 480 mg to about 520 mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage container may be from about 120 mg to about 200 mg (e.g., from about 136 mg to about 184 mg, from about 144 mg to about 176 mg, from about 152 mg to about 168 mg, or from about 156 mg to about 154 mg).

[0122]

[0140] In another example, the egg white protein formulation comprises, consists essentially of, or consists of about 40% to about 60% by weight of dry egg white protein powder, about 25% to about 45% by weight of a first diluent (such as pregelatinized starch), about 10% to about 20% by weight of a second diluent (such as microcrystalline cellulose), about 0.25% to about 0.75% by weight of a lubricant (such as magnesium stearate), and about 0.25% to about 0.75% by weight of a lubricant (such as colloidal silicon dioxide). In some embodiments, the egg white protein formulation comprises about 32% to about 48% by weight of egg white protein. The egg white protein formulation may be packaged in a dosage container (such as a capsule, e.g., a size 00 capsule) having an intended egg white protein dose (i.e., label claim) of 200 mg. The dosage container may contain about 480 mg to about 520 mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage container may be from about 150 mg to about 250 mg (e.g., from about 170 mg to about 230 mg, from about 180 mg to about 220 mg, from about 190 mg to about 210 mg, or from about 195 mg to about 205 mg).

[0123]

[0141] In another example, the egg white protein formulation comprises, consists essentially of, or consists of about 40% to about 60% by weight of dry egg white protein powder, about 25% to about 45% by weight of pregelatinized starch, about 10% to about 20% by weight of microcrystalline cellulose, about 0.25% to about 0.75% by weight of magnesium stearate, and about 0.25% to about 0.75% by weight of colloidal silicon dioxide. In some embodiments, the egg white protein formulation comprises about 32% to about 48% by weight of egg white protein. The egg white protein formulation may be packaged in a dosage container (such as a capsule, e.g., a size 00 capsule) having an intended egg white protein dose (i.e., label claim) of 200 mg. The dosage container may contain about 480 mg to about 520 mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage container may be from about 150 mg to about 250 mg (e.g., from about 170 mg to about 230 mg, from about 180 mg to about 220 mg, from about 190 mg to about 210 mg, or from about 195 mg to about 205 mg).

[0124]

[0142] In another example, the egg white protein formulation comprises, consists essentially of, or consists of about 50% to about 70% by weight of dry egg white protein powder, about 15% to about 35% by weight of a first diluent (such as pregelatinized starch), about 10% to about 20% by weight of a second diluent (such as microcrystalline cellulose), about 0.25% to about 0.75% by weight of a lubricant (such as magnesium stearate), and about 0.25% to about 0.75% by weight of a lubricant (such as colloidal silicon dioxide). In some embodiments, the egg white protein formulation comprises about 40% to about 56% by weight of egg white protein. The egg white protein formulation may be packaged in a dosage container (such as a capsule, e.g., a size 00 capsule) having an intended egg white protein dose (i.e., label claim) of 240 mg. The dosage container may contain about 480 mg to about 520 mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage container may be from about 180 mg to about 300 mg (e.g., from about 204 mg to about 276 mg, from about 216 mg to about 264 mg, from about 228 mg to about 252 mg, or from about 234 mg to about 246 mg).

[0125]

[0143] In another example, the egg white protein formulation comprises, consists essentially of, or consists of about 50% to about 70% by weight of dry egg white protein powder, about 15% to about 35% by weight of pregelatinized starch, about 10% to about 20% by weight of microcrystalline cellulose, about 0.25% to about 0.75% by weight of magnesium stearate, and about 0.25% to about 0.75% by weight of colloidal silicon dioxide. In some embodiments, the egg white protein formulation comprises about 40% to about 56% by weight of egg white protein. The egg white protein formulation may be packaged in a dosage container (such as a capsule, e.g., a size 00 capsule) having an intended egg white protein dose (i.e., label claim) of 240 mg. The dosage container may contain about 480 mg to about 520 mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage container may be from about 180 mg to about 300 mg (e.g., from about 204 mg to about 276 mg, from about 216 mg to about 264 mg, from about 228 mg to about 252 mg, or from about 234 mg to about 246 mg).

[0126]

[0144] In another example, the egg white protein formulation comprises, consists essentially of, or consists of about 60% to about 85% by weight of dry egg white protein powder, about 5% to about 25% by weight of a first diluent (such as pregelatinized starch), about 10% to about 20% by weight of a second diluent (such as microcrystalline cellulose), about 0.25% to about 0.75% by weight of a lubricant (such as magnesium stearate), and about 0.25% to about 0.75% by weight of a lubricant (such as colloidal silicon dioxide). In some embodiments, the egg white protein formulation comprises about 48% to about 68% by weight of egg white protein. The egg white protein formulation may be packaged in a dosage container (such as a capsule, e.g., size 00 capsule, or sachet) having an intended egg white protein dose (i.e., label claim) of 300 mg. The dosage container may contain about 480 mg to about 520 mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage container may be from about 225 mg to about 375 mg (e.g., from about 255 mg to about 345 mg, from about 270 mg to about 330 mg, from about 285 mg to about 315 mg, or from about 292 mg to about 308 mg).

[0127]

[0145] In another example, the egg white protein formulation comprises, consists essentially of, or consists of about 60% to about 85% by weight of dry egg white protein powder, about 5% to about 25% by weight of pregelatinized starch, about 10% to about 20% by weight of microcrystalline cellulose, about 0.25% to about 0.75% by weight of magnesium stearate, and about 0.25% to about 0.75% by weight of colloidal silicon dioxide. In some embodiments, the egg white protein formulation comprises about 48% to about 68% by weight of egg white protein. The egg white protein formulation may be packaged in a dosage container (such as a capsule, e.g., size 00 capsule, or sachet) having an intended egg white protein dose (i.e., label claim) of 300 mg. The dosage container may contain about 480 mg to about 520 mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage container may be from about 225 mg to about 375 mg (e.g., from about 255 mg to about 345 mg, from about 270 mg to about 330 mg, from about 285 mg to about 315 mg, or from about 292 mg to about 308 mg).

[0128]

[0146] The egg white protein preparation, in one embodiment, comprises the major egg white allergens Gal d1, Gal d2, Gal d3, and Gal d4. The amount of each individual allergen can be expressed as a percentage of the total protein in the egg white protein preparation. In one example, a dose of an egg white protein preparation with a label claim of 300 mg contains about 300 mg of egg white protein and 78% Gal d2, meaning that the dose contains about 234 mg of Gal d2. It is understood in the art that egg white proteins can be heavily glycosylated. Thus, the mass of an egg white allergen refers to the mass of the isolated protein, which may include a substantial portion of carbohydrates. Ensuring that an egg white protein preparation has a consistent and known amount of the major egg white allergen is important to ensure the suitability of the composition for use in oral immunotherapy of egg allergy.

[0129]

[0147] In some embodiments, the egg white protein preparation comprises about 5% to about 20% Gal d1, for example, about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 5% to about 15%, or about 10% to about 20% Gal d1, relative to the total mass of egg white protein.

[0130]

[0148] In some embodiments, the egg white protein preparation contains about 45% to about 90% Gal d2, for example, about 45% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 45% to about 60%, about 60% to about 80%, or about 70% to about 90% Gal d2, relative to the total mass of egg white protein.

[0131]

[0149] In some embodiments, the egg white protein preparation contains about 1% to about 20% Gal d3, for example, about 1% to about 5%, about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, or about 5% to about 15% Gal d3, relative to the total mass of egg white protein.

[0132]

[0150] In some embodiments, the egg white protein preparation contains about 0.1% to about 10% Gal d4, for example, about 0.1% to about 3%, about 3% to about 6%, about 6% to about 9%, or about 7% to about 10% Gal d4, relative to the total mass of egg white protein.

[0133] Method for making egg white protein preparations

[0151] The manufacturing method of an egg white protein formulation may affect the batch uniformity of a manufactured formulation lot, the content uniformity of a batch of a dosage container, or the deliverability of the egg white protein formulation from the container. The dose of egg white protein administered during the course of oral immunotherapy can have a wide range (e.g., from about 0.2 mg to about 300 mg), and dosing consistency is important to reduce adverse reactions associated with the treatment, so different dosage formulation manufacturing methods were designed to produce dosage containers with consistent doses.

[0134]

[0152] To produce a consistent dose, the specific method of making the formulation may vary between doses. Process development efforts to optimize blend uniformity and content uniformity were focused on low doses (e.g., doses less than about 3 mg), and process development efforts to ensure proper formulation flowability were focused on high doses (e.g., about 300 mg). Processes for intermediate dose strengths were also developed to properly balance the desired properties of the final product.

[0135]

[0153] The methods described herein also overcome challenges presented in scale-up manufacturing processes. Ensuring batch uniformity at larger formulation quantities can be difficult, and the methods described herein overcome these challenges. This manufacturing method can be used even when producing egg white protein formulation lots of about 5 kg or more (e.g., about 5 kg to about 100 kg, e.g., about 5 kg to about 7 kg, about 7 kg to about 10 kg, about 10 kg to about 15 kg, about 15 kg to about 25 kg, about 25 kg to about 50 kg, or about 50 kg to about 100 kg).

[0136]

[0154] Manufacturing steps such as mixing (e.g., using a tumble blender or conical mill), passing one or more powders or mixtures through a mesh screen, and / or serial dilution when diluents are added to the mixture are used during the manufacturing process. The particular steps used may depend on the egg white protein concentration in the formulation, or the label claim of the dosage form (i.e., the intended amount of egg white protein formulation for the dosage form). Generally, one or more high shear mixing steps (higher shear compared to other steps in the manufacturing process) are incorporated into the manufacturing process sequence to uniformly disperse the dry egg white protein powder in the mixture. Higher shear mixing also disperses soft, low-density aggregates of colloidal silicon dioxide, if present in the formulation, typically observed in colloidal silicon dioxide raw materials. The use of higher power mixing is not intended to reduce the primary particle size of any of the mixture components.

[0137]

[0155] After a formulation is manufactured, the quality of one or more of the allergenic egg white proteins (e.g., ovalbumin, ovomucoid, ovotransferrin, and / or lysozyme) in the formulation and / or the blend uniformity of the formulation can be evaluated. The quality of one or more allergenic proteins can be evaluated, for example, by characterizing one or more of ovalbumin, ovomucoid, ovotransferrin, and / or lysozyme (e.g., by measuring the amount or relative amount of one or more of the proteins, or the potency or relative potency of one or more of the proteins). The blend uniformity of the formulation can be evaluated by measuring the protein content of the formulation, which can be compared to the intended protein content of the formulation (e.g., label claim or intended concentration). For example, measuring the blend uniformity of the formulation can include identifying the intended protein content associated with the formulation, measuring the protein content in a sample from the formulation, and comparing the measured protein content to the intended protein content. Blend uniformity can be measured using multiple samples from a formulation (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more samples) and the average blend uniformity and / or relative standard deviation (RSD) can be measured. By using the manufacturing methods described herein, the blend uniformity RSD of multiple samples from an egg white protein formulation is about 15% or less (e.g., about 0.5% to about 15%, or about 1% to about 10%), or about 5% or less (e.g., about 1% to about 5%, or about 2% to about 5%).

[0138]

[0156] The manufactured formulation can be packaged in a predetermined amount in a dosage container, such as a capsule or sachet. The amount of formulation added to each dosage container is intended to provide an amount of egg white protein equal to the label claim of the dosage container. Due to real-world variations occurring during manufacturing and packaging, there may be some deviation from the label claim and the actual amount of egg white protein in each individual dosage container. Thus, the content uniformity of a manufactured lot of dosage containers can be measured. The content uniformity is based on the deliverable protein content of the formulation from the dosage container. The dosage container is associated with an intended amount of egg white protein in the dosage container. Thus, measuring the content uniformity of a plurality of dosage containers includes identifying an intended protein content associated with the plurality of dosage containers, measuring the deliverable protein content from a sample taken from the plurality of dosage containers (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more dosage containers), and comparing the measured protein content to the intended protein content. When multiple dosage container samples are taken from multiple dose containers, the average content uniformity and / or content uniformity RSD can be determined. Using the manufacturing methods described herein, the content uniformity RSD of the multiple dosage containers is about 12% or less (e.g., about 4% to about 12%, or about 5% to about 12%).

[0139] Low-Dose Manufacturing Process

[0157] Blend uniformity and content uniformity are generally considered to be at greater risk for low dose strengths, which correspond to lower percentages of drug substance in the blend. These low doses are generally no more than about 12 mg of egg white protein in a dosage container, although larger doses can be produced using similar methodologies. Formulation manufacturing processes generally produce formulations with about 0.05% to about 4% egg white protein by weight (about 0.1% to about 0.7% by weight, about 0.7% to about 1.5% by weight, about 1.5% to about 2.5% by weight, or about 2.5% to about 4% by weight egg white protein).

[0140]

[0158] Manufactured egg white protein formulations generally include two diluents (e.g., pregelatinized starch and microcrystalline cellulose). One or more of the diluents are mixed stepwise with the egg white protein powder (i.e., serial dilution of the egg white protein powder), where some (but not all) of the egg white protein powder and diluent are mixed before adding an additional amount of the diluent to the mixture. The blend uniformity of the fully manufactured egg white protein formulation is improved by passing the first mixture containing the egg white protein powder and the first portion of the diluent through a mesh screen before further serial dilution.

[0141]

[0159] 1, in step 102, the dried egg white protein powder is combined with a first amount of a first diluent (e.g., pregelatinized starch). The dried egg white protein powder and the first amount of the first diluent can be added to any suitable container, such as a bottle or bag, and mixed together before proceeding to the next step. Mixing can be performed, for example, by shaking the container or by using an impeller, a blender (such as a tumble blender), or any other suitable device.

[0142]

[0160] Once the dried egg white protein powder is combined with the first amount of the first diluent to form a first mixture, the first mixture is passed through a mesh screen (see step 104 in FIG. 1). The size of the mesh screen is generally about 250 μm to about 850 μm (e.g., about 300 μm to about 710 μm, or about 425 μm to about 600 μm). The size of the mesh screen refers to the average size of the openings in the mesh screen.

[0143]

[0161] Once the first mixture passes through the mesh screen, the mixture can be further diluted by mixing the first mixture with an additional amount of the first diluent to form a second mixture, as shown in step 106 of FIG. 1. Alternatively, the first mixture and a portion of the additional amount of the first diluent can be co-sieved through a mesh screen corresponding to step 104 of FIG. 1. The first mixture and the additional amount of the first diluent can be mixed by shaking the combined components or by using an impeller, a blender (such as a tumble blender), or any other suitable device. The additional amount of the first diluent can be added in stages to continuously dilute the mixture, as shown by arrow 108 of FIG. 1. For example, the first mixture can be mixed with a second amount of the first diluent in multiple repeated substeps. The substeps can include adding a portion of the second amount of the first diluent to the first mixture and mixing a portion of the second amount of the first diluent and the first mixture. Another portion of the second amount of the first diluent can then be added, which is further mixed. These dilution substeps can be performed 1, 2, 3, 4, 5 or more times until the desired amount of the first diluent is mixed with the dry egg white protein powder. Optionally, the mixture can be passed through a mesh screen before adding an additional portion of the second amount of the first diluent.

[0144]

[0162] Alternatively, the first mixture and the second amount of the first diluent can be mixed together by continuously mixing the first mixture while the second amount of the first diluent is being added to the mixture, i.e., instead of incrementally adding the second amount of the first diluent to the first mixture to form the second mixture, the mixture is mixed simultaneously as the second amount of the first diluent is added.

[0145]

[0163] Once the second mixture is formed, it can be mixed with a second diluent (e.g., microcrystalline cellulose) to form a third mixture, as shown in step 110 of FIG. 1. The second mixture and the second diluent can be mixed by shaking the combined components or by using an impeller, a blender (such as a tumble blender), or any other suitable device. The mixture is also subjected to mixing at a higher shear force than that used to form the previous mixture, as shown in step 112. The high shear force mixing may occur after the second mixture is first mixed with the second diluent, or may be the mixing force for mixing the second mixture with the second diluent. This can be performed, for example, using a conical mill (which can be equipped, for example, with a round mill impeller or a square mill impeller) or other suitable device. In some embodiments, the second mixture and the second diluent are mixed using higher shear mixing to first form a third mixture, and in some embodiments, the second mixture and the second diluent are premixed to form a third mixture, and then the third mixture is subjected to high shear mixing. The high shear mixing increases the uniform dispersion of the dried egg white protein powder in the formulation and disperses any soft, low density aggregates that may have formed. The use of higher shear mixing is not intended to reduce the primary particle size of any of the mixture components, but only to disaggregate particle aggregates.

[0146]

[0164] As shown in step 114, a lubricant (e.g., magnesium stearate) is added to the mixture to form the egg white protein formulation. The lubricant can be added to any of the mixtures during the manufacturing process, but at some point is mixed with the mixture containing the dry egg white protein powder, the first diluent, and the second diluent. For example, the lubricant can be mixed with the third mixture before, after, or during the high shear mixing step. An additional amount of one or more diluents (e.g., a first diluent such as pregelatinized starch) may be co-added to the mixture with the lubricant. In addition, the third mixture may be mixed with the lubricant (and optionally an additional amount of the first diluent) and then mixed with an additional amount of the third mixture. In an exemplary embodiment, the third mixture, an additional amount of the first diluent, and the lubricant are mixed, passed through a mesh screen, and then mixed with an additional amount of the third mixture to form the egg white protein formulation. In some embodiments, additional amounts of lubricant and first diluent are mixed (and optionally, the mixture is passed through a mesh screen) before being mixed with the mixture containing the egg white protein.

[0147]

[0165] Optionally, the egg white protein preparation can be passed through a mesh screen, the size of which is generally about 250 μm to about 850 μm (e.g., about 300 μm to about 710 μm, or about 425 μm to about 600 μm).

[0148]

[0166] Batches of egg white protein preparations produced using these methods can be assayed for blend uniformity or quality attributes as further described herein. In some embodiments, the methods further comprise assaying the egg white protein preparation for blend uniformity and / or quality attributes.

[0149]

[0167] In an exemplary method of making an egg white protein formulation, the method includes (a) mixing dried egg white protein powder with a first amount of a first diluent (such as pregelatinized starch) to form a first mixture; (b) combining and sieving the first mixture with a second portion of the first diluent through a mesh screen; (c) after steps (a) and (b), mixing the first mixture with the second portion of the first diluent to form a second mixture; (d) mixing the second mixture with a third portion of the first diluent to form a third mixture; and (e) mixing the third mixture with a fourth portion of the first diluent to form a fourth mixture. (f) mixing the fourth mixture with a fifth portion of the first diluent to form a fifth mixture, (g) mixing the fifth mixture with a second diluent (such as microcrystalline cellulose) to form a sixth mixture, (h) mixing a first portion of the sixth mixture with a lubricant (such as magnesium stearate) and optionally the sixth portion of the first diluent to form a seventh mixture, (j) passing the seventh mixture through a mesh screen, and (k) mixing the screened mixture of (j) with a second portion of the sixth mixture to form the egg white protein formulation. Step (g) may include two mixing sub-steps, one mixing sub-step being at a higher shear force than the other mixing sub-step. In an exemplary embodiment, step (g) optionally includes two substeps, the first substep being at a lower shear force than the second substep (e.g., using a tumble blender), and the second substep being at a higher shear force than the first substep (e.g., using a conical mill). This method of making the formulation is particularly useful for formulations used to make low-dose dosage containers containing the formulation, such as about 0.1 mg to about 12 mg doses (e.g., about 0.2 mg, about 1 mg, about 3 mg, about 6 mg, or about 12 mg dosage containers, or any dosages therebetween). The formulation may have, for example, about 0.05% to about 2.5% by weight of egg white protein. In some embodiments, the formulation is free or substantially free of lubricants. In some embodiments, the formulation is free or substantially free of colloidal silicon dioxide).Batches of egg white protein preparations produced using these methods can be assayed for blend uniformity or quality attributes as further described herein. In some embodiments, the methods further comprise assaying the egg white protein preparation for blend uniformity and / or quality attributes.

[0150]

[0168] An exemplary method for producing a low-dose formulation of egg white protein is shown in FIG. 12A. In step 402, a dry egg white protein powder (preferably characterized to ensure total protein level and specific allergen level) is mixed with a first amount of a first diluent (such as pregelatinized starch) to form a first mixture. In some embodiments, step 402 further comprises characterizing the dry egg white protein powder before mixing with the first amount of the first diluent. In step 404, the first mixture is sieved together with a second amount of the first diluent. In step 406, the combined sieved first mixture and the second amount of the first diluent are mixed to form a second mixture. Step 408 allows for incremental addition of additional amounts of the first diluent to the second mixture. Step 408 may include one, two, three, or more sub-steps of adding and mixing additional amounts of the first diluent. These steps are performed sequentially to allow for greater content uniformity. In step 410, the second mixture is mixed with a second diluent (such as microcrystalline cellulose) to form a third mixture. In step 412, the third mixture is mixed by high shear mixing (e.g., by a conical mill) to disperse particle agglomerates. In step 414, a portion of the dispersed third mixture is mixed with a lubricant to form a fourth mixture. Step 414 may also include mixing a portion of the dispersed third mixture with an additional amount of the first diluent. In step 416, the fourth mixture is passed through a mesh screen. In step 418, the screened fourth mixture and an additional portion of the dispersed third mixture are combined and then mixed in step 420 to form the egg white protein preparation. Batches of egg white protein preparations produced using these methods can be assayed for blend uniformity or quality attributes as further described herein. In some embodiments, the method further includes a step of assaying the egg white protein preparation for blend uniformity and / or quality attributes.

[0151]

[0169] In another exemplary method of making an egg white protein formulation, the method includes (a) mixing dried egg white protein powder with a first amount of a first diluent (such as pregelatinized starch) to form a first mixture; (b) combining and sieving the first mixture with a second portion of the first diluent through a mesh screen; (c) mixing the first mixture with the second portion of the first diluent to form a second mixture; (d) mixing the second mixture with a third portion of the first diluent to form a third mixture; (e) mixing the third mixture with a fourth portion of the first diluent to form a fourth mixture; and (f) mixing the fourth mixture with a fourth portion of the first diluent to form a fourth mixture. (g) mixing the fifth mixture with a second diluent (such as microcrystalline cellulose) to form a sixth mixture; (h) mixing the sixth mixture, optionally using a higher shear force than that used to mix the fifth mixture with the second diluent in step (g); (i) mixing a lubricant (such as magnesium stearate) and a sixth portion of the first diluent to form a seventh mixture, optionally passing the seventh mixture through a mesh screen; and (k) mixing the seventh mixture with the sixth mixture to form the egg white protein formulation. This method of making the formulation is particularly useful for formulations used to make low dose dosage containers containing a dosage of about 0.1 mg to about 12 mg (e.g., about 0.2 mg, about 1 mg, about 3 mg, about 6 mg, or about 12 mg, or any dosage in between). The formulation may have, for example, from about 0.05% to about 2.5% by weight egg white protein. In some embodiments, the formulation is free or substantially free of lubricants. In some embodiments, the formulation is free or substantially free of colloidal silicon dioxide.In some embodiments, the dried egg white protein powder (i.e., before being formulated) and / or the egg white protein formulations produced using these methods (i.e., after being formulated) can be assayed for blend uniformity or quality attributes further described herein, such as measuring the concentration or profile of one or more of ovalbumin, ovomucoid, lysozyme, and / or ovotransferrin, or the total protein content of the composition, using an HPLC assay (e.g., RP-HPLC) or an ELISA assay. The formulated composition may be packaged, for example, in a capsule or sachet.

[0152]

[0170] An exemplary method of producing a low-dose formulation of egg white protein is shown in FIG. 12B. In step 422, dry egg white protein powder is mixed with a first portion of a first diluent (such as pregelatinized starch) to form a first mixture. In step 424, the first mixture is combined and sieved through a mesh screen with a second portion of the first diluent, and the combined first mixture and the second portion of the first diluent are mixed in step 426 to form a second mixture. In step 428, the second mixture is mixed with a third portion of the first diluent (e.g., using a blender such as a tumble blender) to form a third mixture. In step 430, the third mixture is mixed with a fourth portion of the first diluent (e.g., using a blender such as a tumble blender) to form a fourth mixture. In step 432, the fourth mixture is mixed with a fifth portion of the first diluent (e.g., using a blender such as a tumble blender) to form a fifth mixture. In step 434, the fifth mixture is mixed with a second diluent (such as microcrystalline cellulose) to form a sixth mixture. Optionally, the sixth mixture is mixed using a higher shear force than that used in step 434, for example, using a conical mill. In step 436, the sixth portion of the first diluent is mixed with a lubricant (such as magnesium stearate) to form a seventh mixture, which is mixed with the sixth mixture in step 438 to form an eighth mixture (e.g., a formulated composition).

[0153]

[0171] In another exemplary method of making an egg white protein formulation, the method includes: (a) mixing dried egg white protein powder with a first amount of a first diluent (such as pregelatinized starch) to form a first mixture; (b) sifting the first mixture through a mesh screen with a second portion of the first diluent and optionally mixing the combined and sifted composition; (c) serially diluting the combined and sifted composition with one or more additional portions (e.g., 1, 2, 3, 4, 5 or more additional portions) of the first diluent and mixing the composition after each addition of the portion to form a third mixture; (d) mixing the third mixture with a second diluent (such as microcrystalline cellulose) to form a fourth mixture; (e) mixing an additional portion of the first diluent with a lubricant (such as magnesium stearate) to form a fifth mixture; and (f) mixing the fourth mixture with the fifth mixture to form a formulated composition. This method of making the formulation is particularly useful for formulations used to make low dose dosage containers containing the formulation, such as about 0.1 mg to about 12 mg doses (e.g., about 0.2 mg, about 1 mg, about 3 mg, about 6 mg, or about 12 mg dosage containers, or any dosages in between). The formulations may have, for example, about 0.05% to about 2.5% by weight egg white protein. In some embodiments, the formulations are free or substantially free of lubricants. In some embodiments, the formulations are free or substantially free of colloidal silicon dioxide). In some embodiments, the dried egg white protein powders (i.e., before being formulated) and / or egg white protein formulations produced using these methods (i.e., after being formulated) can be assayed for blend uniformity or quality attributes further described herein, such as using an HPLC assay (e.g., RP-HPLC) or an ELISA assay to measure the concentration or profile of one or more of ovalbumin, ovomucoid, lysozyme, and / or ovotransferrin, or the total protein content of the composition. The formulated composition may be packaged, for example, in a capsule or sachet.

[0154]

[0172] An exemplary method of producing a low-dose formulation of egg white protein is shown in FIG. 12C. In step 440, dry egg white protein powder is mixed with a first portion of a first diluent (such as pregelatinized starch) to form a first mixture. In step 442, the first mixture is combined with a second portion of the first diluent and sieved through a mesh screen. In step 444, the first mixture is mixed with a second amount of the first diluent to form a second mixture. In step 446, the second mixture is serially diluted by mixing the second mixture with one or more additional portions of the first diluent to form a third mixture. The serial dilution may be performed, for example, in 1, 2, 3, 4, 5, or more substeps, where a portion of the first diluent is mixed with the composition in each substep before adding an additional portion of the first diluent. In step 448, the third mixture is mixed with a second diluent (such as microcrystalline cellulose) to form a fourth mixture. In step 450, another portion of the first diluent is mixed with a lubricant (such as magnesium stearate) to form a fifth mixture, which is mixed with the fourth mixture in step 452 to form the formulated composition.

[0155]

[0173] The egg white protein preparation can then be packaged into packaging, for example, in a capsule or sachet, to obtain a desired dosage of egg white protein in the dosage container. About 100 mg to about 1000 mg (e.g., about 100 mg to about 250 mg, about 250 mg to about 400 mg, about 400 mg to about 600 mg, or about 600 mg to about 1000 mg) of the egg white protein preparation can be included in the dosage container. By way of example, in some embodiments, about 180 mg of the egg white protein preparation is included in the container (e.g., a capsule) to obtain a desired amount of the egg white protein preparation in the dosage container.

[0156]

[0174] Once the egg white protein formulation is packaged into dosage containers, the dosage containers can be assayed for content uniformity.

[0157] Mid-range production process

[0175] When manufacturing a formulation for a particular dose (such as about 3 mg to about 300 mg in a dosage container, although larger doses can be produced using similar methodology), blend uniformity and content uniformity are balanced with the flowability of the egg white protein formulation. The formulation manufacturing process generally produces a formulation having about 1.5% to about 60% by weight of egg white protein (e.g., about 1.5% to about 3% by weight, about 3% to about 8% by weight, about 8% to about 15% by weight, about 15% to about 30% by weight, or about 30% to about 60% by weight of egg white protein).

[0158]

[0176] Manufactured egg white protein formulations generally include two diluents (e.g., pregelatinized starch and microcrystalline cellulose), a lubricant, and an optional glidant (such as colloidal silicon dioxide). However, for some medium strength dosage forms, colloidal silicon dioxide is not included in the formulation.

[0159]

[0177] As shown in Figure 2, in step 202, the dried egg white protein powder is mixed with a first amount of a first diluent (e.g., pregelatinized starch) to form a first mixture. Optionally, a lubricant (such as colloidal silicon dioxide) can also be mixed with the dried egg white protein powder in this step. Mixing is preferably performed using higher shear forces to disperse particle agglomerates. This can be done, for example, using a conical mill mixer (which can be configured to have a round mill impeller or a square mill impeller).

[0160]

[0178] The first mixture can be further diluted by mixing the first mixture with an additional amount of a first diluent to form a second mixture, as shown in step 204 of Figure 2. The first mixture and the additional amount of the first diluent can be mixed by shaking the combined components or by using an impeller, a blender (such as a tumble blender), or any other suitable device.

[0161]

[0179] As shown in step 206 of FIG. 2, the second mixture can be mixed with a second diluent (e.g., microcrystalline cellulose) to form a third mixture. Optionally, an additional amount of the first diluent is added to the second mixture or the third mixture. The additional amount of the first diluent can be mixed with the second mixture before mixing with the second diluent. Alternatively, the additional amount of the first diluent can be mixed together with the second mixture and the second diluent. The second mixture and the second diluent (and optionally the additional amount of the first diluent) can be mixed by shaking the combined components or by using an impeller, a blender (such as a tumble blender), or any other suitable device.

[0162]

[0180] The third mixture is also subjected to mixing at a higher shear than that used to form the second mixture, as shown in step 208, which disperses particle agglomerates in the mixture. The high shear mixing may occur after the second mixture is first mixed with the second diluent, or may be a mixing force to mix the second mixture with the second diluent. This can be done, for example, using a conical mill (which may be equipped, for example, with a round mill impeller or a square mill impeller) or other suitable equipment. In some embodiments, the second mixture and the second diluent are mixed using higher shear mixing to first form the third mixture, and in some embodiments, the second mixture and the second diluent are premixed to form the third mixture, after which the third mixture is subjected to high shear mixing. The high shear mixing increases the uniform dispersion of the dried egg white protein powder in the formulation and disperses any soft, low density agglomerates that may have formed. The use of higher shear mixing is not intended to reduce the primary particle size of any of the mixture components, but only to break up particle agglomerates.

[0163]

[0181] As shown in step 210, a lubricant (e.g., magnesium stearate) is added to the mixture to form the egg white protein formulation. The lubricant can be added to any of the mixtures during the manufacturing process, but at some points is mixed with the mixture containing the dry egg white protein powder, the first diluent, and the second diluent. For example, the lubricant can be mixed with the third mixture before, after, or during the high shear mixing step. An additional amount of one or more diluents (e.g., a first diluent such as pregelatinized starch) may be co-added to the mixture with the lubricant. In some embodiments, the third mixture may be mixed with the lubricant (and optionally an additional amount of the first diluent) and then mixed with an additional amount of the third mixture. In an exemplary embodiment, the third mixture, an additional amount of the first diluent, and the lubricant are mixed, passed through a mesh screen, and then mixed with an additional amount of the third mixture to form the egg white protein formulation.

[0164]

[0182] Optionally, the egg white protein preparation can be passed through a mesh screen, the size of which is generally about 250 μm to about 850 μm (e.g., about 300 μm to about 710 μm, or about 425 μm to about 600 μm).

[0165]

[0183] An exemplary method for producing an intermediate dose formulation of egg white protein is shown in FIG. 13A. In step 502, dry egg white protein powder (preferably characterized to ensure total protein level and specific allergen level) is mixed with a first amount of a first diluent (such as pregelatinized starch) to form a first mixture. Step 502 may also include mixing with a lubricant (such as colloidal silicon dioxide) to improve flowability. In some embodiments, step 502 further includes characterizing the dry egg white protein powder before mixing with the first amount of the first diluent. In step 504, the first mixture is mixed with a second amount of the first diluent to form a second mixture. The mixing step of 504 may include two sub-steps, one sub-step being at a higher shear force than the other sub-step. In an exemplary embodiment of the method, step 504 optionally includes two substeps, the first substep being at a higher shear than the second substep (such as using a conical mill) and the second substep being at a lower shear (such as using a tumble blender). Step 506 is optional and allows for the addition of an additional amount of the first diluent. In an exemplary embodiment, step 506 includes mixing the second mixture with an additional amount of the first diluent. In another exemplary embodiment, step 506 is skipped. In step 508, the second mixture is mixed with a second diluent (such as microcrystalline cellulose) to form a third mixture. Step 508 may also include mixing the second mixture with an additional amount of the first diluent. In step 510, the third mixture is mixed by a high shear mixing step (e.g., a conical mill) to disperse particle agglomerates. In step 512, a portion of the dispersed third mixture is mixed with a lubricant (such as magnesium stearate) to form a fourth mixture. Step 512 optionally includes mixing the third mixture with an additional amount of the first diluent. In step 514, the fourth mixture is passed through a mesh screen. In step 516, the screened fourth mixture is combined with an additional amount of the dispersed third mixture and then mixed in step 518 to form the egg white protein formulation.

[0166]

[0184] Batches of egg white protein preparations produced using these methods can be assayed for blend uniformity or quality attributes as further described herein. In some embodiments, the methods further comprise assaying the egg white protein preparation for blend uniformity and / or quality attributes.

[0167]

[0185] In some methods of making an egg white protein preparation, the method includes (a) mixing dry egg white protein powder with a first amount of a first diluent to form a first mixture, (b) mixing the first mixture with a second portion of the first diluent to form a second mixture, (c) mixing the second mixture with a third portion of the first diluent to form a third mixture, (d) mixing the third mixture with the second diluent to form a fourth mixture, (e) mixing the fourth mixture with a fourth portion of the first diluent and a lubricant to form a fifth mixture, (f) passing the fifth mixture through a mesh screen, and (g) mixing the screened mixture of (f) with an additional amount of the fourth mixture to form the egg white protein preparation. In step (b), the mixing step may include two substeps, one substep being at a higher shear force than the other substep. In an exemplary embodiment, step (b) comprises two sub-steps, the first sub-step comprises mixing at higher shear than the second sub-step (e.g., using a conical mill), and the second sub-step comprises mixing in a blender (e.g., using a tumble blender). In step (d), the mixing step may comprise two sub-steps, one sub-step at higher shear than the other sub-step. In an exemplary embodiment, step (d) comprises two sub-steps, the first sub-step comprises mixing at lower shear than the second sub-step. In some embodiments, the formulation is free or substantially free of a lubricant. In some embodiments, the formulation is free or substantially free of colloidal silicon dioxide. In some embodiments, the dried egg white protein powder (i.e., before being formulated) and / or the egg white protein formulations produced using these methods (i.e., after being formulated) can be assayed for blend uniformity or quality characteristics further described herein, such as measuring the concentration or profile of one or more of ovalbumin, ovomucoid, lysozyme, and / or ovotransferrin, or the total protein content of the composition, using an HPLC assay (e.g., RP-HPLC) or an ELISA assay.The formulated composition may be packaged, for example, in a capsule or sachet.

[0168]

[0186] In another method of making an egg white protein formulation, the method includes (a) mixing dry egg white protein powder with a first amount of a first diluent (such as pregelatinized starch) to form a first mixture; (b) mixing the first mixture with a second portion of the first diluent to form a second mixture, optionally mixing the second mixture at a shear force lower than the shear force used to mix the first mixture with the second portion of the first diluent; and (c) mixing the second mixture with a third portion of the first diluent to form a third mixture. (d) mixing the third mixture with a second diluent (such as microcrystalline cellulose) to form a fourth mixture, optionally mixing the fourth mixture with a higher shear force than that used to mix the third mixture with the second diluent; (e) mixing a fourth portion of the first diluent with a lubricant (such as magnesium stearate) to form a fifth mixture, optionally passing the fifth mixture through a mesh screen; and (f) mixing the fourth mixture with the fifth mixture. In some embodiments, the formulation is free or substantially free of a lubricant. In some embodiments, the formulation is free or substantially free of colloidal silicon dioxide. In some embodiments, the dried egg white protein powder (i.e., before being formulated) and / or the egg white protein formulations produced using these methods (i.e., after being formulated) can be assayed for blend uniformity or quality attributes further described herein, such as measuring the concentration or profile of one or more of ovalbumin, ovomucoid, lysozyme, and / or ovotransferrin, or the total protein content of the composition, using an HPLC assay (e.g., RP-HPLC) or an ELISA assay. The formulated composition may be packaged, for example, in a capsule or sachet.

[0169]

[0187] FIG. 13B shows another exemplary method of making an egg white protein formulation. Step 520 includes mixing a dry egg white protein powder with a first portion of a first diluent (such as pregelatinized starch) to form a first mixture. Step 522 includes mixing the first mixture with a second portion of the first diluent to form a second mixture. This step may include two or more sub-steps with different mixing shear forces. For example, the first mixture may be mixed with the second portion of the first diluent using a first mixing shear force (e.g., using a conical mill) to form a second mixture, and the second mixture may be further mixed using a second shear force (e.g., using a blender such as a tumble blender) that is lower than the first shear force. Step 524 includes mixing the second mixture with a third portion of the first diluent to form a third mixture. In step 526, the third mixture is mixed with a second diluent (such as microcrystalline cellulose) to form a fourth mixture. This step may include two or more sub-steps with different mixing shear forces. For example, the third mixture may be mixed with the second diluent using a first mixing shear force (e.g., using a blender such as a tumble blender) to form a fourth mixture, and the fourth mixture may be further mixed using a second shear force higher than the first shear force (e.g., using a conical mill). In step 528, a fourth portion of the first diluent is mixed with a lubricant (such as magnesium stearate) to form a fifth mixture, which is optionally passed through a mesh screen. In step 530, the fourth mixture is mixed with the fifth mixture to form the egg white protein formulation.

[0170]

[0188] In another method of making an egg white protein formulation, the method includes (a) mixing dry egg white protein powder with a first amount of a first diluent (such as pregelatinized starch) and a lubricant (such as colloidal silicon dioxide) to form a first mixture; (b) mixing the first mixture with a second portion of the first diluent to form a second mixture, optionally mixing the second mixture at a shear force lower than the shear force used to mix the first mixture with the second portion of the first diluent; and (c) mixing the second mixture with a third portion of the first diluent to form a third mixture. (d) mixing the third mixture with a second diluent (such as microcrystalline cellulose) and a fourth portion of the first diluent to form a fourth mixture, optionally mixing the fourth mixture with a higher shear force than the shear force used to mix the third mixture with the second diluent, (e) mixing a fifth portion of the first diluent with a lubricant (such as magnesium stearate) to form a fifth mixture, optionally passing the fifth mixture through a mesh screen, and (f) mixing the fourth mixture with the fifth mixture. In some embodiments, the dried egg white protein powder (i.e., before being formulated) and / or the egg white protein formulations (i.e., after being formulated) produced using these methods can be assayed for blend uniformity or quality characteristics further described herein, such as measuring the concentration or profile of one or more of ovalbumin, ovomucoid, lysozyme, and / or ovotransferrin, or the total protein content of the composition, using an HPLC assay (e.g., RP-HPLC) or an ELISA assay. The formulated composition may be packaged, for example, in a capsule or sachet.

[0171]

[0189] FIG. 13C shows another exemplary method of making an egg white protein formulation. Step 532 includes mixing the dried egg white protein powder with a first portion of a first diluent (such as pregelatinized starch) and a lubricant (such as colloidal silicon dioxide) to form a first mixture. In step 534, the first mixture is mixed with a second portion of the first diluent to form a second mixture. This step may include two or more sub-steps with different mixing shear forces. For example, the first mixture may be mixed with the second portion of the first diluent using a first mixing shear force (e.g., using a conical mill) to form a second mixture, and the second mixture may be further mixed using a second shear force (e.g., using a blender such as a tumble blender) that is lower than the first shear force. In step 536, the second mixture is mixed with a third portion of the first diluent and a second diluent (such as microcrystalline cellulose) to form a third mixture. This step may include two or more sub-steps with different mixing shear forces. For example, the third mixture may be mixed with the first diluent using a first mixing shear force (e.g., using a blender such as a tumble blender) to form a fourth mixture, and the fourth mixture may be further mixed using a second shear force higher than the first shear force (e.g., using a conical mill). In step 538, the fourth portion of the first diluent is mixed with the first portion with a lubricant (such as magnesium stearate) to form a fifth mixture, which is optionally passed through a mesh screen. In step 540, the fifth mixture is mixed with the fourth mixture.

[0172]

[0190] The egg white protein preparation can then be packaged into packaging, for example, in a capsule or sachet, to obtain a desired dosage of egg white protein in the dosage container. About 100 mg to about 1000 mg (e.g., about 100 mg to about 250 mg, about 250 mg to about 400 mg, about 400 mg to about 600 mg, or about 600 mg to about 1000 mg) of the egg white protein preparation can be included in the dosage container. By way of example, in some embodiments, about 180 mg of the egg white protein preparation is included in the container (e.g., a capsule) to obtain a desired amount of the egg white protein preparation in the dosage container. In some embodiments, about 500 mg of the egg white protein preparation is included in the container (e.g., a capsule) to obtain a desired amount of the egg white protein preparation in the dosage container.

[0173]

[0191] Once the egg white protein formulation is packaged into dosage containers, the dosage containers can be assayed for content uniformity.

[0174] High-Dose Manufacturing Process

[0192] The manufacture of egg white protein formulations for use at a particular strength dose (such as about 100 mg or more) is designed to prioritize the flowability of the formulation. Egg white protein powder may adhere to packaging materials and limit the deliverability of the formulation from the container. To increase flowability, formulations are generally manufactured with a lubricant (such as colloidal silicon dioxide) in addition to one or more diluents (such as pregelatinized starch and / or microcrystalline cellulose) and lubricants (such as magnesium stearate). In some embodiments, the egg white protein formulation has about 50% to about 80% by weight (e.g., about 50% to about 60% by weight, about 60% to about 70% by weight, or about 70% to about 80% by weight) of egg white protein.

[0175]

[0193] To produce the egg white protein formulation, the dry egg white protein powder, the first diluent (such as pregelatinized starch), and the lubricant (such as colloidal silicon dioxide) are mixed together as shown in step 302 of Figure 3. The components can be mixed by shaking the combined components or by using an impeller, blender (such as a tumble blender), or any other suitable device.

[0176]

[0194] The first mixture is then mixed with a second diluent (such as microcrystalline cellulose) to form a second mixture, as shown in step 304. The first mixture and the second diluent can be mixed using higher shear forces than were used to mix the egg white protein powder with the first diluent and lubricant. The higher shear forces can break up particle agglomerates within the mixture.

[0177]

[0195] The second mixture is also mixed with a lubricant to form the egg white protein formulation, as shown in step 306. Optionally, an additional amount of the first diluent can be mixed with the second mixture before, after, or at the same time that the lubricant is mixed with the second mixture. In some embodiments, the second mixture may be mixed with the lubricant (and optionally an additional amount of the first diluent) and then mixed with an additional amount of the second mixture. In an exemplary embodiment, the second mixture, the additional amount of the first diluent, and the lubricant are mixed, passed through a mesh screen, and then mixed with an additional amount of the second mixture to form the egg white protein formulation.

[0178]

[0196] Optionally, the egg white protein preparation can be passed through a mesh screen, the size of which is generally about 250 μm to about 850 μm (e.g., about 300 μm to about 710 μm, or about 425 μm to about 600 μm).

[0179]

[0197] An exemplary method for producing a high-dose formulation of egg white protein is shown in FIG. 14A. In step 602, a dry egg white protein powder (preferably characterized to ensure total protein level and specific allergen level) is mixed with a first amount of a first diluent (such as pregelatinized starch) and a lubricant (such as colloidal silicon dioxide) to form a first mixture. In some embodiments, step 602 further includes characterizing the dry egg white protein powder before mixing with the first amount of the first diluent. In step 604, the first mixture is then mixed with a second diluent to form a second mixture, which is then mixed using a high shear mixing step (such as a conical mill) to disperse particle agglomerates. In step 606, a portion of the dispersed second mixture is mixed with a lubricant to form a third mixture. Step 606 may also include mixing the second mixture with an additional amount of the first diluent. In step 608, the third mixture is passed through a mesh screen. In step 610, the screened third mixture and a second amount of the dispersed second mixture are combined and then mixed in step 612 to form the egg white protein preparation.

[0180]

[0198] In some methods of making an egg white protein formulation, the method includes (a) mixing dry egg white protein powder with a first amount of a first diluent (such as pregelatinized starch) to form a first mixture; (b) mixing the first mixture with a second diluent (such as microcrystalline cellulose), optionally at a higher shear force than was used to form the first mixture in step (a); (c) mixing a second portion of the first diluent with a lubricant (such as magnesium stearate) to form a third mixture, optionally passing the third mixture through a mesh screen; and (d) mixing the second mixture with the third mixture. In some embodiments, the dried egg white protein powder (i.e., before being formulated) and / or the egg white protein formulations produced using these methods (i.e., after being formulated) can be assayed for blend uniformity or quality attributes further described herein, such as measuring the concentration or profile of one or more of ovalbumin, ovomucoid, lysozyme, and / or ovotransferrin, or the total protein content of the composition, using an HPLC assay (e.g., RP-HPLC) or an ELISA assay. The formulated composition may be packaged, for example, in a capsule or sachet.

[0181]

[0199] Another exemplary method for producing a high-dose formulation of egg white protein is shown in FIG. 14B. In step 614, dry egg white protein powder is mixed with a first portion of a first diluent (such as pregelatinized starch) and a lubricant (such as colloidal silicon dioxide) to form a first mixture. In step 616, the first mixture is mixed with a second diluent (such as microcrystalline cellulose) to form a second mixture. In some embodiments, the dry egg white protein powder is mixed with the first portion of the first diluent and the lubricant in step 614 to form a first mixture, the first mixture is mixed with the second diluent at higher shear (e.g., using a conical mill), and the first mixture is mixed using a blender, such as a tumble blender. In step 618, a second portion of the first diluent is mixed with a lubricant (such as magnesium stearate) to form a third mixture, which is optionally passed through a mesh screen. In step 620, the second mixture is mixed with the third mixture.

[0182]

[0200] Batches of egg white protein preparations produced using these methods can be assayed for blend uniformity or quality attributes as further described herein. In some embodiments, the methods further comprise assaying the egg white protein preparation for blend uniformity and / or quality attributes.

[0183]

[0201] The egg white protein preparation can then be packaged into packaging, for example, in a capsule or sachet, to obtain a desired dosage of egg white protein in the dosage container. About 100 mg to about 1000 mg (e.g., about 100 mg to about 250 mg, about 250 mg to about 400 mg, about 400 mg to about 600 mg, or about 600 mg to about 1000 mg) of the egg white protein preparation can be included in the dosage container. By way of example, in some embodiments, about 500 mg of the egg white protein preparation is included in the container (e.g., a capsule) to obtain a desired amount of the egg white protein preparation in the dosage container.

[0184]

[0202] Once the egg white protein formulation is packaged into dosage containers, the dosage containers can be assayed for content uniformity.

[0185] Quality Control Process

[0203] The dosage forms and egg white protein formulations described herein can be used for oral immunotherapy, where increasing doses are administered to patients allergic to one or more egg proteins to desensitize the patient to those proteins. Because allergic reactions to allergenic proteins can be severe and life-threatening, it is desirable that the dose administered is well controlled, quantitatively and qualitatively. To ensure quality control of the products administered to patients, various parameters of the dried egg white protein powder, the egg white protein formulation, and the dosage containers (and dosage container lots) containing the egg white protein formulation can be monitored. Such quality control processes can include characterizing one or more allergenic egg white proteins (ovomucoid, ovalbumin, ovotransferrin, and / or lysozyme), measuring protein content, measuring blend uniformity of the egg white protein formulation, measuring content uniformity or deliverable mass of a lot of dosage containers containing the egg white protein formulation, or measuring water activity of the dried egg white protein powder or the egg white protein formulation. Quality control methods can be included in the manufacturing process to ensure consistent production of the egg white protein preparation or dosage containers.

[0186]

[0204] Allergenic proteins in egg white include Gal d1 (ovomucoid), Gal d2 (ovalbumin), Gal d3 (ovotransferrin), and Gal d4 (lysozyme). One or more (or all) of these proteins in an egg white protein preparation or dried egg white protein powder can be characterized using the methods described herein. Characterization of one or more allergenic egg white proteins may include measuring the presence of one or more proteins, the amount or relative amount of one or more immunodominant egg white proteins, or the potency or relative potency of one or more immunodominant egg white proteins.

[0187]

[0205] One method of characterizing ovomucoid, ovalbumin, ovotransferrin and / or lysozyme involves analyzing dried egg white protein powder or egg white protein preparations using high performance liquid chromatography (HPLC). Examples of HPLC techniques include size exclusion chromatography (SEC-HPLC) and reverse phase HPLC (RP-HPLC). RP-HPLC, for example, sufficiently separates all four allergenic egg white proteins qualitatively or quantitatively for analysis. An HPLC profile can be obtained and peaks representing ovomucoid, ovalbumin, ovotransferrin and / or lysozyme can be identified or compared to reference standards for qualitative assessment.

[0188]

[0206] Characterizing ovomucoid, ovalbumin, ovotransferrin and / or lysozyme may include measuring the presence of one or more of ovomucoid, ovalbumin, ovotransferrin and / or lysozyme in the dried egg white protein powder (drug substance) and / or egg white protein formulation (pharmaceutical product). This can be done, for example, by polyacrylamide gel electrophoresis (PAGE), such as SDS-PAGE, immunoblot, or HPLC (such as SEC-HPLC or RP-HPLC). Allergenic proteins generally elute from the HPLC column at consistent time points, and peaks can be identified using a reference standard (such as purified commercially available ovomucoid, ovalbumin, ovotransferrin, or lysozyme). Allergenic proteins in the dried egg white protein powder or egg white protein formulation can be identified using an HPLC profile, for example based on retention time. The HPLC profile can also be qualitatively compared to the reference standard to measure the consistency of the tested dried egg white protein powder or egg white protein formulation.

[0189]

[0207] Characterizing ovomucoid, ovalbumin, ovotransferrin and / or lysozyme can include quantifying the amount of ovomucoid, ovalbumin, ovotransferrin and / or lysozyme in an egg white protein preparation or dried egg white protein powder. Proteins can be suitably quantified using HPLC (e.g., SEC-HPLC or RP-HPLC) methods, for example, by measuring the area of ​​the peaks in the HPLC profile. The concentration of ovomucoid, ovalbumin, ovotransferrin and / or lysozyme in an egg white protein preparation or dried egg white powder can be measured using this method, for example, by comparing the protein peaks associated with ovomucoid, ovalbumin, ovotransferrin and / or lysozyme to a reference protein peak. Another example of characterizing proteins includes measuring the relative amount of ovomucoid, ovalbumin, ovotransferrin and / or lysozyme using an HPLC profile. The relative amount can be compared to the total protein in the egg white protein preparation or dried egg white protein preparation (e.g., by comparing to the total area of ​​all protein peaks) or to the total amount of ovomucoid, ovalbumin, ovotransferrin and / or lysozyme. The HPLC profile is established by measuring the absorbance of light, such as at a light wavelength of about 210 nm to about 280 nm, as the proteins elute from the column. The peak area due to the protein in the HPLC profile correlates with the amount of the protein in the assayed sample. However, the ratio of peak areas between different proteins may not necessarily reflect the weight ratio of the different proteins due to differences in the absorbance of the proteins. The weight ratio of the different proteins can be established by calibrating the peak areas of the investigated proteins to mass. Thus, the relative amounts of the proteins may be, for example, the relative peak areas from the HPLC profile or the relative weights of the proteins. Any measure of the relative amounts of the allergenic proteins can be used as a quality control process to ensure lot-to-lot consistency.

[0190]

[0208] One or more of ovomucoid, ovalbumin, ovotransferrin and / or lysozyme can also be characterized to determine the potency or relative potency of the allergenic egg white proteins in the dried egg white protein powder or egg white protein formulation (relative to the potency of the proteins in a reference sample). The potency or relative potency of the allergenic proteins may vary due to denaturation of the allergenic proteins, and measuring the potency or relative potency of the allergenic proteins in the dried egg white protein powder or egg white protein formulation may be useful to monitor the quality or consistency of the powder and / or formulation. In some embodiments, the potency of one or more egg white proteins in the dried egg white protein powder or egg white protein formulation is measured in vitro. In some embodiments, a single allergenic protein (e.g., ovomucoid) is used to represent the potency or relative potency of the powder or formulation. Potency can be measured, for example, by the EC measured by a potency assay. 50 or EC 50 The potency of one or more allergenic egg white proteins may be reported as a ratio (i.e., relative potency). Exemplary immunoassay techniques that can be used to measure the potency of one or more allergenic egg white proteins include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoblot, surface plasmon resonance (SPR), or multiplexed immunoassay. Another method for measuring the potency of ovalbumin, ovotransferrin, ovomucoid, or lysozyme in an egg white protein formulation may include a cell-based assay (e.g., basophil histamine release assay). See, for example, Santos et al., Basophil activation test: food challenge in a test tube or specialist research tool?, Clinical and Translational Allergy, vol. 6, no. 10, pp. 1-9 (2016).

[0191]

[0209] The reporter molecule used in the potency assay binds to the assayed allergenic protein with high specificity and is generally an antibody. The antibody may be a monoclonal or polyclonal antibody. In some embodiments, the antibody is an IgG or IgE antibody. The antibody used to measure potency or relative potency specifically binds to the assayed protein (e.g., ovalbumin, ovotransferrin, ovomucoid, or lysozyme). An antibody pool comprising antibodies that specifically bind to two or more different proteins can also be used to simultaneously measure the potency or relative potency of two or more assay proteins. For example, the pool may comprise two or more of an antibody that specifically binds to ovalbumin, an antibody that specifically binds to ovotransferrin, an antibody that specifically binds to ovomucoid, and / or an antibody that specifically binds to lysozyme. The antibody pool may be a pool of separately purified antibodies mixed together or may be derived from animals immunized with whole egg protein or egg white protein. The same antibody or pool of antibodies should be used for the test and reference samples when measuring relative potency.

[0192]

[0210] The protein content of dried egg white protein powder or egg white protein formulations can be measured to monitor the quality during the manufacturing process, either before or after packaging in a dosage form. Protein content can be reported as a concentration (e.g., weight percentage of the composition) or with reference to a target (i.e., intended) amount of protein (e.g., X% of the target dose). The protein content of dried egg white protein powder is generally measured to measure how much dried egg white protein powder should be included during the manufacturing process to obtain the intended egg white protein concentration in the manufactured egg white protein formulation. Protein content can also be used to measure blend uniformity in an egg white protein formulation, or content uniformity in a lot of manufactured dosage containers containing the egg white protein formulation. Exemplary methods for measuring protein content include light absorbance, Lowry assay, Bradford assay, combustion assay, bicinchoninic acid (BCA) assay, HPLC (such as SEC-HPLC, RP-HPLC, or any other chromatographic method that can quantitatively measure the amount of protein), or any other suitable quantitative protein assay.

[0193]

[0211] The deliverable mass of the egg white protein formulation from a dosage container can be measured on an individual dosage container basis or as a sample of dosage containers taken from a manufactured lot. The deliverable mass of a lot of dosage containers can be determined by sampling a number of dosage containers selected from the lot (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more dosage containers). A composition is deliverable from a container when the composition can be retrieved from the container by pouring the composition from the container, shaking the container, or tapping the container. The deliverable mass can be determined by weighing the contents of a dosage form deliverable from a dosage container under normal use. A composition that can only be removed from a container by inserting a mechanical device into the container or by adding a fluid (such as a compressed gas or liquid) into the container is not deliverable from the container.

[0194]

[0212] The water activity of the dried egg white protein powder or egg white protein formulation can be measured. Water activity can be measured by use of a water activity meter such as the AQUALAB 4TEV. The dried egg protein formulation should be relatively dry to avoid microbial growth and / or deterioration of the allergenic egg white protein.

[0195] Oral immunotherapy methods

[0213] The patient treated using the oral immunotherapy method described herein is a human individual with egg allergy, preferably hen's egg allergy. The egg allergy can be an allergy to raw egg white protein or an allergy to cooked egg protein (either cooked egg white protein or cooked whole egg protein). The cooked egg protein can be baked.

[0196]

[0214] The egg white protein preparations and dosage forms described herein can use oral immunotherapy to treat human patients who are allergic to eggs (preferably chicken eggs). Egg allergy can be an allergy to raw egg white protein or an allergy to cooked egg protein (either cooked egg white protein, such as baked egg white protein or baked whole egg protein, or cooked whole egg protein). The most abundant allergenic proteins found in egg white are ovalbumin, ovomucoid, ovotransferrin, and lysozyme, and patients can be allergic to one or more of these allergenic proteins.

[0197]

[0215] Methods for diagnosing egg allergy are known in the art. For example, patients can be diagnosed using skin prick tests (SPT), egg white protein specific IgE (ew-IgE) levels, oral food challenges, or association of one or more allergic responses with egg protein ingestion. For example, in some embodiments, patients may have a serum egg allergy level of about 0.35 kU. A / L or more, approximately 0.7kU A / L, approximately 3.5kU A / L or more, approximately 5kUA / L or more, or about 7kU A The patient has a serum ew-IgE level of 1000 mg / L or more. The serum ew-IgE level can be measured using a quantitative immunoassay. Quantitative immunoassays are known in the art and can include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), alkaline phosphatase immunoassay autoanalyzers such as the IMMULITE® system (Siemens Healthcare Diagnostics, Erlangen, Germany), radioallergosorbent test (RAST) such as the ImmunoCAP® system (Thermo Fisher Scientific / Phadia, Uppsala, Sweden), or fluorescent enzyme immunoassay autoanalyzers. Fluorescent enzyme immunoassay autoanalyzers are the preferred method.

[0198]

[0216] The patient is generally about 4 years of age or older, for example, about 4 to 26 years of age.

[0199]

[0217] A baseline maximum tolerated dose or baseline cumulative tolerated dose of raw egg white protein or cooked egg protein (either cooked egg white protein or cooked whole egg protein, such as baked egg white protein or baked whole egg protein) can be determined for the patient prior to the initiation of treatment with an oral food challenge. Oral food challenge involves oral administration of a series of doses of an allergenic composition (e.g., egg white protein or cooked egg protein, such as baked egg white protein or baked whole egg protein) until a moderate or severe adverse reaction associated with the administered egg white protein is elicited. Each dose administered is spaced apart by a period of time (e.g., about 20 minutes to about 30 minutes) and the patient is monitored for adverse events. An exemplary food challenge is a double-blind, placebo-controlled food challenge (DBPCFC), as described in Sampson et al., Standardizing double blind, placebo controlled oral food challenges: American Academy of Allergy, Asthma & Immunology European Academy of Allergy and Clinical Immunology PRACTALL consensus report, J. Allergy Clin. Immunol., vol. 130, no. 6, pp. 1260-74 (2012).

[0200]

[0218] Oral food loading for raw egg white protein to determine the maximum tolerated dose or cumulative tolerated dose of raw egg white protein may include oral administration of a series of increasing amounts of raw egg white protein. The dose may range, for example, from about 1 mg to about 2000 mg, or from about 1 mg to about 300 mg. The patient does not need to take all the doses of the oral food loading, since the oral food loading is generally terminated after the patient suffers from a moderate or severe allergenic adverse event. An exemplary set of raw egg white doses for oral food loading is shown in Table 1. For the exemplary oral food loading doses shown in Table 1, if the maximum dose of egg white protein tolerated by the patient is 30 mg (maximum tolerated dose), the cumulative tolerated dose is 44 mg of egg white protein.

[0201] [Table 1]

[0202]

[0219] In some embodiments, the maximum dose of raw egg white protein tolerated by a patient at the initiation of treatment (i.e., maximum tolerated dose) is about 2000 mg or less, about 1000 mg or less, about 600 mg or less, about 300 mg or less, about 100 mg or less, about 30 mg or less, about 10 mg or less, or about 3 mg or less. In some embodiments, the maximum cumulative dose of raw egg white protein tolerated by a patient at the initiation of treatment (i.e., maximum tolerated dose) is about 444 mg or less, about 144 mg or less, about 44 mg or less, about 14 mg or less, or about 4 mg or less. Because some patients are highly sensitive to raw egg white protein, in order to reduce the risk of severe adverse events over the course of treatment, in some embodiments, the maximum tolerated dose is about 0.2 mg or more of raw egg white protein, about 0.4 mg or more of raw egg white protein, about 0.8 mg or more of raw egg white protein, about 1.0 mg or more of raw egg white protein, about 1.2 mg or more of raw egg white protein, or about 2 mg or more of raw egg white protein. In some embodiments, the cumulative tolerated dose is about 0.2 mg or more of raw egg white protein, about 0.6 mg or more of raw egg white protein, about 1.4 mg or more of raw egg white protein, about 2.4 mg or more of raw egg white protein, about 2.6 mg or more of raw egg white protein, about 4.4 mg or more of raw egg white protein, or about 4.6 mg or more of raw egg white protein.

[0203]

[0220] Oral food challenge with cooked or baked egg protein can also be used to determine the maximum tolerated dose of cooked or baked egg protein. Cooked or baked egg protein can be from cooked or baked egg white or cooked or baked whole egg (i.e., egg white and egg yolk). In oral food challenge, there is a range of doses of cooked egg protein (e.g., cooked or fried in baked food products such as bread, muffin, cookie, or cake). An exemplary set of cooked or baked egg doses for oral food challenge is shown in Table 2, which has the dose of cooked whole egg protein in baked muffin. In some embodiments, the baked egg protein or baked egg white protein is heated at about 176°C to about 260°C (e.g., about 176°C to about 232°C, about 176°C to about 220°C, or about 176°C to about 205°C) for about 20 minutes to about 60 minutes (e.g., about 20 minutes to about 45 minutes, about 20 minutes to about 35 minutes, or about 20 minutes to about 30 minutes).

[0204] [Table 2]

[0205]

[0221] Some patients treated with oral immunotherapy described herein can tolerate cooked egg protein, while some patients cannot tolerate any or large amounts of cooked or baked egg protein. In some embodiments, the maximum dose of cooked egg protein or baked egg protein tolerated by the patient at the start of treatment (i.e., maximum tolerated dose) is about 625 mg or less, about 500 mg or less, about 250 mg or less, or about 125 mg or less. In some embodiments, the maximum cumulative dose of cooked egg protein or baked egg protein tolerated by the patient at the start of treatment (i.e., maximum tolerated dose) is about 2000 mg or less, about 1375 mg or less, about 875 mg or less, about 375 mg or less, or about 125 mg or less.

[0206] Oral Immunotherapy Treatment Schedule

[0222] According to an oral immunotherapy (OIT) schedule, a patient is treated for egg allergy by orally administering to the patient multiple doses of a pharmaceutical composition comprising egg white protein. The OIT schedule typically includes a dose escalation period, which is followed by a maintenance period. Optionally, the OIT schedule further includes an initial titration period that occurs before the start of the dose escalation period.

[0207]

[0223] The doses may be provided in one or more dosage containers (e.g., capsules or sachets) that contain the pharmaceutical composition (also referred to herein as "egg white protein formulations"). The dosage containers are typically not ingested in their entirety, but are generally opened prior to administration, and the pharmaceutical composition contained therein is ingested orally. The pharmaceutical composition may be mixed with a food vehicle that is consumed by the patient. Exemplary food vehicles include applesauce, pudding, oatmeal, beverages (e.g., shakes), or any other suitable food product to be mixed with the pharmaceutical composition.

[0208] Dose escalation period

[0224] The dose escalation period of the OIT schedule involves orally administering a series of increasing daily doses of egg white protein to the patient. The egg white protein is generally raw, but may be pasteurized or otherwise processed in a manner that does not affect the presentation of protein epitopes. The egg white protein is included within an egg white protein preparation, which may be produced, for example, according to the methods described herein. A dose of egg white protein is preferably administered to the patient daily, but a dose may be skipped, delayed, or a portion of a dose may be delayed if the patient experiences one or more adverse events associated with administration of egg white protein, or a co-factor associated with increased sensitivity to an allergen not associated with administration of egg white protein (such as atopic disease flare, inflammation, illness, menstruation, or unintentional exposure to a food to which the patient is allergic).

[0209]

[0225] A series of increasing doses of egg white protein generally ranges from about 1 mg to about 300 mg. A given dose is administered to the patient daily (unless adjustments are made) for at least two weeks before the dose is titrated to a higher dose. Titration to a higher dose may take longer than two weeks, for example due to adverse events related to egg white protein or some other concurrent factor related to increased sensitivity to allergens not related to egg white protein, which justifies a delay in increasing the dose. The dose administered during the dose escalation period is escalated only if the patient tolerates the previous dose. Thus, in some cases, the same dose may be administered to the patient for more than two weeks, such as for three weeks or more, or four weeks or more. To ensure that the dose is not titrated too rapidly, the dose escalation period generally lasts for about 20 weeks or more, for example, from about 20 weeks to about 44 weeks.

[0210]

[0226] The dose escalation period may include 4, 5, 6, 7, 8, 9, or 10 or more different doses administered to the subject, which may range from about 1 mg to about 300 mg. The different doses may be spaced apart within this dose range, but need not be evenly spaced apart. Exemplary doses administered to the subject may include about 1 mg, about 3 mg, about 6 mg, about 12 mg, about 20 mg, about 40 mg, about 80 mg, about 120 mg, about 160 mg, about 200 mg, about 240 mg, and about 300 mg of egg white protein. In some embodiments, the dose escalation period includes administration of at least 1 mg and 300 mg of egg white protein. In some embodiments, the maximum dose administered to the patient during the dose escalation period is 300 mg of egg white protein.

[0211]

[0227] The escalating dose is preferably administered at a clinical site (e.g., in a doctor's office, hospital, or other facility that allows immediate medical response when moderate or severe allergic adverse events occur).For example, if a patient completes a two-week period of daily administration of an 80 mg dose of egg white protein, a 120 mg dose of egg white protein can be administered at a clinical site.If the patient tolerates the escalating dose, the next dose at that dose can be administered to the subject outside of a clinical site, such as by the patient self-administering at home.

[0212] Retention period

[0228] Following the dose escalation period, the OIT schedule includes a maintenance period. The maintenance period involves administering a dose of egg white protein to the patient for a period of time, and is intended to maintain a state of desensitization after completion of treatment. Generally, the maintenance period is about 12 weeks or longer in length, but does not need to have a defined end point. In some embodiments, the maintenance dose is administered daily, although dosage adjustments can be made as described herein.

[0213]

[0229] The egg white protein dose administered during the maintenance period (i.e., the "maintenance dose") is usually the maximum tolerated dose obtained by the patient during the dose escalation period, although the dose may be reduced under certain circumstances. For example, if a patient successfully completes a dose escalation period involving administering 300 mg of egg white protein, the maintenance dose, which is typically 300 mg of egg white protein, may be lowered. For example, if a patient experiences one or more adverse events associated with administration of egg white protein, or concurrent factors associated with increased sensitivity to allergens not associated with administration of egg white protein (such as atopic disease flare, inflammation, illness, or menstruation), the maintenance dose may be reduced, skipped, delayed, or partially delayed.

[0214] Early Titration

[0230] Optionally, the initial titration phase precedes the dose escalation period in the OIT schedule. The initial titration phase involves orally administering to the patient a series of increasing doses of egg white protein per day. The doses are spaced apart for a period of time, generally about 20 minutes or more (e.g., about 20 minutes to about 60 minutes, or about 20 minutes to about 30 minutes). The temporal separation allows the patient to be monitored for adverse events related to the egg white protein before the next dose is administered. Generally, the initial titration phase is performed in a clinical setting.

[0215]

[0231] The egg white protein dose administered to the subject can be, for example, about 0.2 mg of egg white protein to about 2 mg of egg white protein. The initial titration phase can include 2, 3, 4, 5, 6 or more doses. Exemplary doses administered to the subject can include about 0.2 mg, about 0.4 mg, about 0.8 mg, about 1.2 mg, and about 2 mg of egg white protein. In additional exemplary embodiments, the doses administered to the subject can include about 0.2 mg, about 0.4 mg, about 0.8 mg, about 1.0 mg, and about 2 mg of egg white protein.

[0216] Dose Adjustment

[0232] The oral immunotherapy schedule can be adjusted slightly if the patient experiences an adverse event associated with the administration of the egg white protein dose (i.e., an allergic reaction to the dose) or a co-factor associated with increased sensitivity to an allergen not associated with the administration of the egg white protein dose. The adverse event associated with the administration of the egg white protein can be a mild allergenic adverse event, a moderate allergenic adverse event, or a severe allergenic adverse event. This dose adjustment can be made during the dose escalation period of the OIT schedule or the maintenance period of the OIT schedule. The dose adjustment can be a reduction in dose, skipping a scheduled dose administration, delaying a portion of the dose, or delaying the dose titration. Adjusting the dose schedule reduces the risk of experiencing a more severe adverse event (if the dose is adjusted in response to an adverse event associated with the administration of the egg white protein) or the risk of experiencing an adverse event or severity of an adverse event (if the dose is adjusted in response to an adverse event not associated with the administration of the egg white protein).

[0217]

[0233] Adverse events associated with administration of egg white protein that may lead to dosage adjustments may be, for example, hypersensitivity, anaphylaxis (e.g., anaphylactic shock), or gastrointestinal symptoms (such as abdominal pain or vomiting). Figure 4 shows an exemplary decision tree for adjusting dosage depending on the severity of adverse events associated with administration of egg white protein.

[0218]

[0234] The cofactor associated with increased sensitivity to an allergen that may result in dosage adjustment may be any event or patient condition that increases the patient's sensitivity to an allergen that occurs simultaneously with administration of the pharmaceutical composition. This may be due to, for example, immunological changes resulting from the cofactor associated with increased sensitivity to an allergen. Exemplary cofactors associated with increased sensitivity to an allergen include atopic disease flare-up (e.g., eczema, asthma, or rhinoconjunctivitis), allergic reactions to allergens other than egg white protein, inflammation (e.g., due to surgery or traumatic injury), illness (such as infection), and menstruation. The cofactor may also be unintentional exposure (e.g., ingestion) to a food to which the patient is allergic. The food to which the patient is allergic may be egg, or any other food to which the patient is allergic, such as milk, peanuts, nuts, wheat, shellfish, or soybeans.

[0219]

[0235] The dose of the pharmaceutical composition can be adjusted by reducing the dose of the pharmaceutical composition. The dose reduction is temporary and can be increased after a period of time, for example, after the passage of a concurrent factor associated with an adverse event or increased sensitivity to the allergen. The dose can be reduced up to about 50%, or to the next available dose lower than 50%, relative to the previously administered dose. In some embodiments, the dose is reduced one or two dose levels below the previously administered dose. In some embodiments, one or more (e.g., a first) reduced dose is administered at the clinical site. Once the dose is reduced, the reduced dose can be administered to the patient for about one week or more (e.g., two weeks or more, about one week to about four weeks, or about one week to about two weeks) before titrating the subsequent dose. The titrated dose is preferably administered at the clinical site so that the patient can be monitored for adverse events associated with administration of the pharmaceutical composition. Administration of the titrated dose should be attempted, but if an allergic reaction results, the reduced dose can be continued to be administered to the patient.

[0220]

[0236] In some embodiments, the dose is adjusted by skipping one or more scheduled dose administrations.For example, if the dose is scheduled to be administered daily, one, two, three or more daily doses can be skipped before resuming administration of the pharmaceutical composition.Administration of the pharmaceutical composition can be resumed at the same dose as previously administered or at a reduced dose.If more than one dose is skipped, it is generally preferred that the next dose administered to the patient is administered at the clinical site.

[0221]

[0237] Adjusting the dosage of the pharmaceutical composition may include delaying administration of a portion of the pharmaceutical composition. For example, a first portion can be administered to the patient as scheduled, and a second portion is administered to the patient at a time prior to the next scheduled dose administration. In some embodiments, the dose is split into two approximately equal portions. The second portion may be administered to the patient about 8 hours to about 16 hours, or about 8 hours to about 12 hours, after administration of the first portion.

[0222]

[0238] Dosage administration can also be adjusted during the ramp-up phase by delaying the scheduled ramp-up of the dose administered to the patient. OIT schedules generally have a preset ramp-up time, such as every 2 weeks. However, if the patient is experiencing adverse events or concurrent factors associated with increased sensitivity to allergens, ramping up the dose level may increase the risk of adverse events or more severe adverse events. Thus, ramp-up can be delayed, for example, until the adverse events or concurrent factors subside. In some embodiments, ramp-up is delayed for about 1 week or more, about 2 weeks or more, or about 3 weeks or more.

[0223]

[0239] In one example, a method of adjusting a dosage of a pharmaceutical composition comprising an egg white protein during oral immunotherapy for egg allergy in a subject comprises orally administering a first dose of the pharmaceutical composition to the patient and orally administering a second dose of the pharmaceutical composition to the patient, wherein if the patient experiences an adverse event associated with administration of the first dose, the second dose is reduced, skipped, or at least a portion of the doses is delayed, wherein the oral immunotherapy comprises (i) a dose escalation period comprising orally administering a series of ascending doses of the egg white protein to the patient, and (ii) a maintenance period comprising orally administering a plurality of maintenance doses comprising the egg white protein to the patient, wherein the method comprises delaying the second dose, wherein the delaying may comprise splitting the second dose into a first portion and a second portion, wherein the first portion is administered according to a predetermined dosing schedule, and wherein if the patient experiences an adverse event associated with administration of the first dose, the second portion is delayed (e.g., by about 8 hours to about 16 hours, or by about 8 hours to about 12 hours) relative to the predetermined dosing schedule. In some embodiments, the adverse event associated with administration of the first dose is a mild allergenic adverse event, a moderate allergenic adverse event, or a severe allergenic adverse event. In some embodiments, the first dose and the second dose are administered to the patient during a dose escalation period, and in some embodiments, the first dose and the second dose are administered to the patient during a maintenance period.

[0224]

[0240] In another example, a method of adjusting a dosage of a pharmaceutical composition comprising egg white protein during oral immunotherapy for egg allergy in a subject comprises orally administering a first dose of the pharmaceutical composition to the patient and orally administering a second dose of the pharmaceutical composition to the patient, wherein if the patient experiences a concomitant factor associated with increased sensitivity to an allergen unrelated to administration of the first dose, the second dose is reduced or skipped, and the oral immunotherapy comprises (i) a dose escalation period comprising orally administering a series of increasing doses of egg white protein to the patient, and (ii) a maintenance period comprising orally administering a plurality of maintenance doses comprising egg white protein to the patient, wherein the concomitant factor associated with increased sensitivity to an allergen is, for example, an atopic disease flare, inflammation, illness, or menstruation. In some embodiments, the first dose and the second dose are administered to the patient during the dose escalation period of oral immunotherapy, and in some embodiments, the first dose and the second dose are administered to the patient during the maintenance period of oral immunotherapy.

[0225] Oral immunotherapy treatment endpoints

[0241] The oral immunotherapy described herein is intended to desensitize patients to egg allergens in order to reduce the risk of adverse allergic events due to accidental exposure to egg white protein. The degree of desensitization to raw egg white protein and / or cooked egg protein (either cooked egg white protein or whole egg protein, such as baked egg white protein or baked whole egg protein) can be measured using a food challenge, as described above with reference to the patient's baseline characteristics. Success of treatment can be indicated by an increase in the maximum tolerated dose or an increase in the cumulative tolerated dose to raw egg white protein or cooked (or baked) egg protein after treatment compared to the start of treatment.

[0226]

[0242] In some embodiments, the patient can tolerate a dose of about 300 mg raw egg white protein, about 600 mg raw egg white protein, about 1000 mg raw egg white protein, or about 2000 mg raw egg white protein after the end of the dose escalation period. In some embodiments, the patient can tolerate a dose of about 300 mg raw egg white protein, about 600 mg raw egg white protein, about 1000 mg raw egg white protein, or about 2000 mg raw egg white protein after the end of the maintenance period. In some embodiments, the patient can tolerate a cumulative dose of about 444 mg raw egg white protein, about 1044 mg raw egg white protein, about 2044 mg raw egg white protein, or about 4044 mg raw egg white protein at the end of the dose escalation period. In some embodiments, the patient can tolerate a cumulative dose of about 444 mg raw egg white protein, about 1044 mg raw egg white protein, about 2044 mg raw egg white protein, or about 4044 mg raw egg white protein at the end of the maintenance period.

[0227]

[0243] In some embodiments, if a patient was unable to tolerate a dose of about 625 mg cooked (or baked) egg protein, about 500 mg cooked (or baked) egg protein, about 250 mg cooked (or baked) egg protein, or about 125 mg cooked (or baked) egg protein at the start of treatment, the patient can tolerate a dose of about 625 mg cooked (or baked) egg protein at the end of the dose escalation period. In some embodiments, if a patient was unable to tolerate a dose of about 625 mg cooked (or baked) egg protein, about 500 mg cooked (or baked) egg protein, about 250 mg cooked (or baked) egg protein, or about 125 mg cooked (or baked) egg protein at the start of treatment, the patient can tolerate a dose of about 625 mg cooked (or baked) egg protein at the end of the maintenance period. In some embodiments, if the patient was unable to tolerate a cumulative dose of about 2000 mg cooked (or baked) egg protein, about 1375 mg cooked (or baked) egg protein, about 875 mg cooked (or baked) egg protein, about 375 mg cooked (or baked) egg protein, or about 125 mg cooked (or baked) egg protein at the start of treatment, the patient can tolerate a cumulative dose of about 2000 mg cooked (or baked) egg protein at the end of the dose escalation period. In some embodiments, if the patient was unable to tolerate a cumulative dose of about 2000 mg cooked (or baked) egg protein, about 1375 mg cooked (or baked) egg protein, about 875 mg cooked (or baked) egg protein, about 375 mg cooked (or baked) egg protein, or about 125 mg cooked (or baked) egg protein at the start of treatment, the patient can tolerate a cumulative dose of about 2000 mg cooked (or baked) egg protein at the end of the maintenance period.

[0228] Exemplary embodiments

[0244] The following embodiments are illustrative and should not be construed as limiting the invention described herein.

[0229]

[0245] Embodiment 1. A method of making an egg white protein preparation, comprising: (a) mixing dried egg white protein powder with a first amount of a first diluent to form a first mixture; (b) passing the first mixture through a mesh screen; (c) after steps (a) and (b), combining the first mixture with a second amount of a first diluent to form a second mixture; (d) mixing the second mixture with a second diluent to form a third mixture; (e) mixing the third mixture with a higher shear force than that used for mixing in step (c); (f) mixing the third mixture with a lubricant to form an egg white protein formulation.

[0230]

[0246] Embodiment 2. The method of embodiment 1, wherein the egg white protein preparation has about 0.05% to about 2.5% by weight of egg white protein.

[0231]

[0247] Embodiment 3. The method of embodiment 1 or 2, wherein the egg white protein preparation has about 0.1% to about 0.7% by weight of egg white protein.

[0232]

[0248] Embodiment 4. The method of any one of embodiments 1-3, wherein step (b) comprises passing at least a portion of the second amount of the first diluent along with the first mixture through a mesh screen.

[0233]

[0249] Embodiment 5. The method of any one of embodiments 1-3, wherein step (b) comprises passing a second amount of the first diluent with the first mixture through a mesh screen.

[0234]

[0250] Embodiment 6. The method of any one of embodiments 1-5, wherein step (c) comprises a plurality of substeps, each substep comprising: (i) adding a portion of the second amount of the first diluent to the first mixture; and (ii) mixing the portion of the second amount of the first diluent and the first mixture.

[0235]

[0251] Embodiment 7. The method of embodiment 6, wherein step (c) comprises three or more substeps.

[0236]

[0252] Embodiment 8. The method of any one of embodiments 1-5, wherein step (c) comprises continuously mixing the first mixture with the second amount of the first diluent when the second amount of the first diluent is added to the first mixture.

[0237]

[0253] Embodiment 9. The method of any one of embodiments 1-8, wherein the first mixture is mixed with a second amount of the first diluent in a tumble blender.

[0238]

[0254] Embodiment 10. The method of any one of embodiments 1-9, wherein the second mixture is mixed with a second diluent in a tumble blender.

[0239]

[0255] Embodiment 11. The method of any one of embodiments 1 to 10, wherein the third mixture is mixed using a conical mill.

[0240]

[0256] Embodiment 12. The method of any one of embodiments 1 to 11, wherein the third mixture is mixed with a lubricant in a tumble blender.

[0241]

[0257] Embodiment 13. The method of any one of embodiments 1-12, wherein a third amount of the first diluent is mixed with the third mixture.

[0242]

[0258] Embodiment 14 The method of embodiment 13, wherein a third amount of the first diluent and a lubricant are combined and mixed with the third mixture.

[0243]

[0259] Embodiment 15. The method of any one of embodiments 1 to 14, comprising mixing the lubricant with an additional portion of the first diluent or the second diluent prior to mixing with the third mixture.

[0244]

[0260] Embodiment 16. The method of embodiment 15, wherein the mixture of the lubricant and the additional portion of the first diluent or the second diluent is passed through a mesh screen before the lubricant is mixed with the third mixture.

[0245]

[0261] Embodiment 17. A method of making an egg white protein preparation, comprising: (a) mixing dried egg white protein powder with a first amount of a first diluent to form a first mixture; (b) combining and screening the first mixture with a second portion of the first diluent through a mesh screen to form a second mixture; (c) serially diluting the second mixture with one or more additional portions of the first diluent to form a third mixture; (d) mixing the third mixture with a second diluent to form a fourth mixture; (e) mixing an additional portion of the first diluent or the second diluent with the lubricant to form a fifth mixture; (f) mixing the fourth mixture with the fifth mixture.

[0246]

[0262] Embodiment 18. The method of embodiment 17, comprising mixing a second mixture prior to step (c).

[0247]

[0263] Embodiment 19. The method of embodiment 17 or 18, comprising, prior to step (e), mixing the fourth mixture with a higher shear force than that used to mix the third mixture with the second diluent.

[0248]

[0264] Embodiment 20. The method of any one of embodiments 17-19, comprising sieving the fifth mixture prior to step (f).

[0249]

[0265] Embodiment 21 The method of any one of embodiments 1 to 20, wherein the egg white protein preparation is substantially free of colloidal silicon dioxide.

[0250]

[0266] Embodiment 22. The method of any one of embodiments 1 to 21, wherein the egg white protein preparation is about 40% to about 70% by weight of the first diluent.

[0251]

[0267] Embodiment 23 The method of any one of embodiments 1 to 22, wherein the egg white protein preparation is about 30% to about 50% by weight of the second diluent.

[0252]

[0268] Embodiment 24. The method of any one of embodiments 1 to 23, wherein the egg white protein preparation is from about 0.1% to about 2% by weight of a lubricant.

[0253]

[0269] Embodiment 25. A method of making an egg white protein preparation, comprising: (a) mixing dried egg white protein powder with a first amount of a first diluent to form a first mixture; (b) mixing a second amount of a first diluent with the first mixture to form a second mixture; (c) mixing the second mixture with a second diluent to form a third mixture; (d) mixing the third mixture with a higher shear force than that used for mixing in step (b); (e) mixing the third mixture with a lubricant to form the egg white protein formulation.

[0254]

[0270] Embodiment 26. The method of embodiment 25, wherein the dried egg white protein powder is mixed with the first amount of the first diluent in step (a) at higher shear forces than those used for mixing in step (b).

[0255]

[0271] Embodiment 27. The method of embodiment 25 or 26, wherein the egg white protein preparation has about 1% to about 70% by weight of egg white protein.

[0256]

[0272] Embodiment 28. The method of any one of embodiments 25-27, wherein the dried egg white protein powder is mixed with the first amount of the first diluent in step (a) using a conical mill.

[0257]

[0273] Embodiment 29. The method of any one of embodiments 25 to 28, wherein step (b) comprises two mixing sub-steps, one mixing sub-step being at a higher shear force than the other mixing sub-step.

[0258]

[0274] Embodiment 30. The method of any one of embodiments 25-29, wherein step (c) further comprises mixing the second mixture with an additional amount of the first diluent prior to mixing with the second diluent to form a third mixture.

[0259]

[0275] Embodiment 31. The method of any one of embodiments 25-29, wherein step (c) further comprises combining the second mixture with additional amounts of the first diluent and the second diluent to form a third mixture.

[0260]

[0276] Embodiment 32. The method of any one of embodiments 25 to 31, wherein the third mixture is mixed in step (d) using a conical mill.

[0261]

[0277] Embodiment 33. The method of any one of embodiments 25-32, wherein the second amount of the first diluent and the first mixture are mixed in a tumble blender.

[0262]

[0278] Embodiment 34. The method of any one of embodiments 25-33, wherein the second mixture is mixed with a second diluent in a tumble blender.

[0263]

[0279] Embodiment 35 The method of any one of embodiments 25-33, wherein a third amount of the first diluent is mixed with the third mixture.

[0264]

[0280] Embodiment 36 The method of embodiment 35, wherein a third amount of the first diluent and a lubricant are combined and mixed with the third mixture.

[0265]

[0281] Embodiment 37. The method of any one of embodiments 25-36, wherein step (e) comprises: (i) mixing a portion of the third mixture with a lubricant; (ii) passing the mixture of (i) through a mesh screen; and (iii) mixing the mixture of (ii) with an additional portion of the third mixture to form the egg white protein preparation.

[0266]

[0282] Embodiment 38. The method of any one of embodiments 25 to 37, wherein the third mixture is mixed with a lubricant in a tumble blender.

[0267]

[0283] Embodiment 39. The method of any one of embodiments 25 to 38, wherein the egg white protein preparation is from about 9% by weight to about 85% by weight of the first diluent.

[0268]

[0284] Embodiment 40. The method of any one of embodiments 25-39, wherein the egg white protein preparation is from about 10% by weight to about 50% by weight of the second diluent.

[0269]

[0285] Embodiment 41 The method of any one of embodiments 25 to 40, wherein the egg white protein preparation is about 10% to about 20% by weight of the second diluent.

[0270]

[0286] Embodiment 42. The method of any one of embodiments 25 to 41, wherein the egg white protein preparation is from about 0.1% by weight to about 2% by weight of a lubricant.

[0271]

[0287] Embodiment 43 The method of any one of embodiments 25 to 42, wherein the egg white protein preparation comprises a lubricant.

[0272]

[0288] Embodiment 44. The method of embodiment 43, wherein the lubricant is mixed with the egg white protein powder and the first amount of the first diluent during or before step (a).

[0273]

[0289] Embodiment 45. The method of embodiment 43 or 44, wherein the lubricant is colloidal silicon dioxide.

[0274]

[0290] Embodiment 46 The method of any one of embodiments 25 to 45, wherein the egg white protein preparation is from about 2% to about 70% by weight egg white protein.

[0275]

[0291] Embodiment 47 The method of any one of embodiments 25 to 44, wherein the egg white protein preparation is substantially free of colloidal silicon dioxide.

[0276]

[0292] Embodiment 48. The method of embodiment 47, wherein the egg white protein preparation is about 1% to about 5% by weight egg white protein.

[0277]

[0293] Embodiment 49. A method of making an egg white protein preparation, comprising: (a) mixing dried egg white protein powder with a first diluent and a lubricant to form a first mixture; (b) mixing a second diluent and the first mixture under a higher shear force than that used for mixing in step (a) to form a second mixture; (c) mixing the second mixture and a lubricant to form the egg white protein preparation.

[0278]

[0294] Embodiment 50. The method of embodiment 49, wherein the egg white protein preparation has about 50% to about 80% egg white protein by weight.

[0279]

[0295] Embodiment 51. The method of embodiment 49 or 50, wherein the dry egg white protein powder, the first diluent, and the lubricant are mixed in a tumble blender.

[0280]

[0296] Embodiment 52. The method of any one of embodiments 49 to 51, wherein the second diluent and the first mixture are mixed in step (b) using a conical mill.

[0281]

[0297] Embodiment 53. The method of any one of embodiments 49-52, wherein the second mixture and the lubricant are mixed in a tumble blender.

[0282]

[0298] Embodiment 54 The method of any one of embodiments 49-53, wherein a second amount of the first diluent is mixed with the second mixture.

[0283]

[0299] Embodiment 55 The method of embodiment 54, wherein a second amount of the first diluent and a lubricant are combined and mixed with the second mixture.

[0284]

[0300] Embodiment 56. The method of any one of embodiments 49 to 55, wherein the lubricant comprises colloidal silicon dioxide.

[0285]

[0301] Embodiment 57. The method of any one of embodiments 49-56, wherein step (c) comprises (i) mixing a portion of the second mixture with a lubricant; (ii) passing the portion of the second mixture and the lubricant through a mesh screen; and (iii) mixing (ii) with an additional portion of the second mixture to form the egg white protein preparation.

[0286]

[0302] Embodiment 58 The method of any one of embodiments 1 to 57, wherein the egg white protein preparation is produced in a lot size of about 5 kg or more.

[0287]

[0303] Embodiment 59. The method of embodiment 58, wherein the egg white protein preparation is produced in a lot size of about 5 kg to about 50 kg.

[0288]

[0304] Embodiment 60 The method of any one of embodiments 1-59, comprising measuring the egg white protein blend uniformity of the egg white protein formulation.

[0289]

[0305] Embodiment 61 The method of any one of embodiments 1-60, wherein the egg white protein preparation has an egg white protein blend uniformity relative standard deviation (RSD) of about 15% or less.

[0290]

[0306] Embodiment 62 The method of any one of embodiments 1 to 61, comprising packaging the egg white protein preparation in a multi-dose container.

[0291]

[0307] Embodiment 63. The method of embodiment 62, wherein the dosage container is a capsule or sachet.

[0292]

[0308] Embodiment 64. The method of embodiment 62 or 63, comprising measuring the egg white protein content uniformity of a plurality of dosage containers.

[0293]

[0309] Embodiment 65. The method of any one of embodiments 62-64, wherein the plurality of dose containers has an egg white protein content uniformity relative standard deviation (RDS) of about 15% or less.

[0294]

[0310] Embodiment 66 The method of any one of embodiments 1 to 65, wherein the first diluent is pregelatinized starch.

[0295]

[0311] Embodiment 67. The method of any one of embodiments 1 to 66, wherein the second diluent is microcrystalline cellulose.

[0296]

[0312] Embodiment 68. The method of any one of embodiments 1 to 67, wherein the lubricant is magnesium stearate.

[0297]

[0313] Embodiment 69. The method of any one of embodiments 1 to 68, wherein the dried egg white protein powder comprises about 50% to about 90% egg white protein by weight.

[0298]

[0314] Embodiment 70. The method of any one of embodiments 1 to 69, wherein forming the dried egg white protein powder comprises spray drying liquid egg white.

[0299]

[0315] Embodiment 71. The method of any one of embodiments 1 to 70, wherein the egg white protein powder has been deglucose depleted.

[0300]

[0316] Embodiment 72. The method of any one of embodiments 1 to 71, wherein the dried egg white powder is pasteurized.

[0301]

[0317] Embodiment 73. The method of any one of embodiments 1 to 72, wherein the dried egg white protein powder is derived from chicken eggs.

[0302]

[0318] Embodiment 74. The method of any one of embodiments 1 to 73, further comprising characterizing ovomucoid, ovalbumin, ovotransferrin, or lysozyme in the dried egg white protein powder.

[0303]

[0319] Embodiment 75 The method of any one of embodiments 1 to 74, further comprising characterizing ovomucoid, ovalbumin, ovotransferrin, or lysozyme in the egg white protein preparation.

[0304]

[0320] Embodiment 76. The method of embodiment 74 or 75, wherein characterizing ovomucoid, ovalbumin, ovotransferrin, or lysozyme comprises obtaining a high performance liquid chromatography (HPLC) profile.

[0305]

[0321] Embodiment 77. The method of embodiment 76, wherein the HPLC profile is a reverse-phase HPLC (RP-HPLC) profile.

[0306]

[0322] Embodiment 78. The method of embodiment 76, wherein the HPLC profile is a size exclusion chromatography HPLC (SEC-HPLC) profile.

[0307]

[0323] Embodiment 79. The method according to any one of embodiments 76 to 78, comprising comparing the obtained HPLC profile with a reference HPLC profile.

[0308]

[0324] Embodiment 80. The method of any one of embodiments 74-79, wherein characterizing the ovomucoid, ovalbumin, ovotransferrin, or lysozyme comprises quantifying the amount of ovomucoid, ovalbumin, ovotransferrin, or lysozyme.

[0309]

[0325] Embodiment 81. The method of embodiment 80, wherein quantifying the amount of ovomucoid, ovalbumin, ovotransferrin, or lysozyme comprises measuring the amount of ovomucoid, ovalbumin, ovotransferrin, or lysozyme relative to total protein in the egg white protein powder or egg white protein preparation.

[0310]

[0326] Embodiment 82. The method of embodiment 80, wherein quantifying the amount of ovomucoid, ovalbumin, ovotransferrin, or lysozyme comprises measuring the amount of ovomucoid, ovalbumin, ovotransferrin, or lysozyme relative to the total amount of ovomucoid, ovalbumin, ovotransferrin, and lysozyme in the egg white protein powder or egg white protein formulation.

[0311]

[0327] Embodiment 83. The method of any one of embodiments 74-82, wherein characterizing the ovomucoid, ovalbumin, ovotransferrin, or lysozyme comprises measuring the potency of ovomucoid, ovalbumin, ovotransferrin, or lysozyme in the egg white protein powder or egg white protein formulation.

[0312]

[0328] Embodiment 84. The method of embodiment 83, wherein the potency of ovomucoid, ovalbumin, ovotransferrin, or lysozyme in the egg white protein powder or egg white protein preparation is measured relative to the potency of ovomucoid, ovalbumin, ovotransferrin, or lysozyme in a reference sample.

[0313]

[0329] Embodiment 85 The method of embodiment 83 or 84, wherein the potency of ovomucoid, ovalbumin, ovotransferrin, or lysozyme is measured using an immunoassay.

[0314]

[0330] Embodiment 86. The method of embodiment 85, wherein the immunoassay comprises the use of one or more of an antibody that specifically binds to ovomucoid, an antibody that specifically binds to ovalbumin, an antibody that specifically binds to ovotransferrin, or an antibody that specifically binds to lysozyme.

[0315]

[0331] Embodiment 87. The method of embodiment 85, wherein the immunoassay comprises the use of a pool of antibodies comprising two or more antibodies selected from the group consisting of an antibody that specifically binds to ovomucoid, an antibody that specifically binds to ovalbumin, an antibody that specifically binds to ovotransferrin, and an antibody that specifically binds to lysozyme.

[0316]

[0332] Embodiment 88. The method of embodiments 86 and 87, wherein the antibody is an IgE antibody or an IgG antibody.

[0317]

[0333] Embodiment 89. The method of any one of embodiments 84 to 88, wherein the potency of ovomucoid, ovalbumin, ovotransferrin, or lysozyme is measured using an enzyme-linked immunosorbent assay (ELISA).

[0318]

[0334] Embodiment 90. An egg white protein preparation produced according to the method of any one of embodiments 1 to 89.

[0319]

[0335] Embodiment 91. An egg white protein formulation comprising a dry egg white protein powder, a first diluent, a second diluent, and a lubricant, wherein the egg white protein formulation is substantially free of colloidal silicon dioxide.

[0320]

[0336] Embodiment 92. The egg white protein preparation of embodiment 91, wherein the egg white protein preparation comprises about 0.1% to about 3.5% by weight of egg white protein.

[0321]

[0337] Embodiment 93. An egg white protein formulation according to embodiment 91 or 92, wherein the first diluent is pregelatinized starch.

[0322]

[0338] Embodiment 94. An egg white protein preparation according to any one of embodiments 91 to 93, wherein the second diluent is microcrystalline cellulose.

[0323]

[0339] Embodiment 95. An egg white protein formulation according to any one of embodiments 91 to 94, wherein the lubricant is magnesium stearate.

[0324]

[0340] Embodiment 96 The egg white protein preparation of any one of embodiments 91-95, wherein the egg white protein preparation consists essentially of egg white protein powder, a first diluent, a second diluent, and a lubricant.

[0325]

[0341] Embodiment 97. A method of treating egg allergy in a patient, comprising: orally administering to the patient multiple doses of a pharmaceutical composition comprising egg white protein according to an oral immunotherapy schedule, the oral immunotherapy schedule comprising: (a) a dose escalation period comprising orally administering to a patient a series of ascending doses of about 1 mg to about 300 mg of egg white protein, where a given dose is administered to the patient for at least 2 weeks before the dose is escalated, and the dose escalation period is about 20 weeks to about 44 weeks in length; (b) a maintenance period comprising orally administering to the patient a number of maintenance doses comprising egg white protein, wherein the maintenance period is about 12 weeks or longer in length.

[0326]

[0342] Embodiment 98. The method of embodiment 97, wherein the patient's serum level of egg white-specific IgE (ew-IgE) is about 7 kUA / L or greater at the start of treatment.

[0327]

[0343] Embodiment 99. The method of embodiment 97, wherein the patient's serum level of egg white-specific IgE (ew-IgE) is about 5 kUA / L or greater at the start of treatment.

[0328]

[0344] Embodiment 100. The method of any one of embodiments 97-99, wherein the series of ascending doses administered to the patient during the dose escalation period comprises at least a 1 mg dose of egg white protein, and at least a 300 mg dose of egg white protein.

[0329]

[0345] Embodiment 101. The method of any one of embodiments 97-100, wherein the series of ascending doses administered to the patient during the dose escalation period comprises at least 10 different doses of egg white protein.

[0330]

[0346] Embodiment 102. The method of any one of embodiments 97-101, wherein the series of ascending doses administered to the patient during the dose escalation period comprises doses of about 1 mg, about 3 mg, about 6 mg, about 12 mg, about 20 mg, about 40 mg, about 80 mg, about 120 mg, about 160 mg, about 200 mg, about 240 mg, and about 300 mg of egg white protein.

[0331]

[0347] Embodiment 103. The method of any one of embodiments 97-102, wherein the dose administered during the dose escalation period is increased only if the patient tolerates the previous dose.

[0332]

[0348] Embodiment 104. The method of any one of embodiments 97-103, wherein the maximum dose administered to the patient during the dose escalation period is about 300 mg of egg white protein.

[0333]

[0349] Embodiment 105. The method of any one of embodiments 97-104, wherein the maintenance dose administered to the patient during the maintenance period is about 300 mg or more of egg white protein.

[0334]

[0350] Embodiment 106. The method of any one of embodiments 97-105, wherein the maintenance dose administered to the patient during the maintenance period is about 300 mg of egg white protein.

[0335]

[0351] Embodiment 107. The method of any one of embodiments 97-106, wherein a maintenance dose is administered to the patient only if the patient tolerates the maximum dose administered to the patient during the dose escalation period.

[0336]

[0352] Embodiment 108. The method of any one of embodiments 97-107, wherein the patient tolerates a dose of about 600 mg of raw egg white protein at the end of the maintenance period.

[0337]

[0353] Embodiment 109. The method of any one of embodiments 97-108, wherein the patient tolerates a dose of about 1000 mg of raw egg white protein at the end of the maintenance period.

[0338]

[0354] Embodiment 110. The method of any one of embodiments 97-109, wherein the patient tolerates a dose of about 2000 mg of raw egg white protein at the end of the maintenance period.

[0339]

[0355] Embodiment 111. The method of any one of embodiments 97-110, wherein the patient tolerates a cumulative dose of about 2000 mg of cooked egg white protein at the end of the maintenance period.

[0340]

[0356] Embodiment 112. The method of any one of embodiments 97-111, wherein the patient tolerates a cumulative dose of about 2000 mg of baked egg white protein at the end of the maintenance period.

[0341]

[0357] Embodiment 113. The method of any one of embodiments 97-112, wherein the patient is unable to tolerate a dose of about 300 mg of raw egg white protein prior to the initiation of treatment.

[0342]

[0358] Embodiment 114. The method of any one of embodiments 97 to 113, wherein the patient is unable to tolerate a cumulative dose of about 2000 mg of cooked egg white protein prior to the initiation of treatment.

[0343]

[0359] Embodiment 115. The method of any one of embodiments 97 to 114, wherein the patient is unable to tolerate a cumulative dose of about 2000 mg of baked egg white protein prior to the initiation of treatment.

[0344]

[0360] Embodiment 116. The method of any one of embodiments 97-113, wherein the patient tolerates a cumulative dose of about 2000 mg of cooked egg white protein prior to the initiation of treatment.

[0345]

[0361] Embodiment 117. The method of any one of embodiments 97 to 113 and 116, wherein the patient tolerates a cumulative dose of about 2000 mg of baked egg white protein prior to the initiation of treatment.

[0346]

[0362] Embodiment 118. The method of any one of embodiments 97-117, wherein the oral immunotherapy schedule includes an initial titration phase prior to a dose escalation period, the initial titration phase comprising orally administering to the patient a series of ascending doses of about 0.2 mg to about 2 mg of egg white protein per day, wherein a single dose of any given dose is administered to the patient, and the doses are spaced at least 15 minutes apart.

[0347]

[0363] Embodiment 119. The method of embodiment 118, wherein the patient is treated according to an oral immunotherapy schedule only if the patient tolerates a dose of about 1.0 mg of raw egg white protein on the first day of treatment.

[0348]

[0364] Embodiment 120. The method of any one of embodiments 97-119, wherein the patient is about 4 years of age or older prior to the initiation of treatment.

[0349]

[0365] Embodiment 121. The method of any one of embodiments 97 to 120, wherein the patient is about 4 years to about 26 years of age prior to the initiation of treatment.

[0350]

[0366] Embodiment 122. A method for adjusting the dosage of a pharmaceutical composition comprising an egg white protein during oral immunotherapy of egg allergy in a subject, the oral immunotherapy comprising: (i) a dose escalation period comprising orally administering to the patient a series of increasing doses of an egg white protein; and (ii) a maintenance period comprising orally administering to the patient a plurality of maintenance doses comprising an egg white protein, the method comprising: orally administering to the patient a first dose of the pharmaceutical composition; orally administering to the patient a second dose of the pharmaceutical composition, wherein if the patient experiences an adverse event associated with administration of the first dose, the second dose is reduced, skipped, or at least a portion of the dose is delayed.

[0351]

[0367] Embodiment 123. The method of embodiment 122, wherein the second dose is divided into a first portion and a second portion, the first portion is administered according to a predetermined administration schedule, and if the patient experiences an adverse event associated with administration of the first dose, the second portion is delayed relative to the predetermined administration schedule.

[0352]

[0368] Embodiment 124. The method of embodiment 123, wherein the second portion is delayed from about 8 hours to about 12 hours after the first portion is administered.

[0353]

[0369] Embodiment 125. The method of embodiment 122, wherein if the patient experiences an adverse event associated with administration of the first dose, the second dose is skipped.

[0354]

[0370] Embodiment 126. The method of embodiment 122, wherein the second dose is reduced relative to the first dose if the patient experiences an adverse event associated with administration of the first dose.

[0355]

[0371] Embodiment 127. The method of embodiment 126, wherein the subsequent doses of the pharmaceutical composition are reduced relative to the first dose for about one week or more before the subsequent doses are titrated up.

[0356]

[0372] Embodiment 128. The method of embodiment 126 or 127, wherein the subsequent doses of the pharmaceutical composition are reduced relative to the first dose for about 1 week to about 2 weeks before attempting to titrate up the subsequent doses.

[0357]

[0373] Embodiment 129. The method of any one of embodiments 126 to 128, wherein the subsequent doses of the pharmaceutical composition are reduced relative to the first dose for about 1 week to about 2 weeks before titrating the subsequent doses.

[0358]

[0374] Embodiment 130. The method of any one of embodiments 122-129, wherein the adverse event associated with administration of the first dose is a mild allergenic adverse event.

[0359]

[0375] Embodiment 131. The method of any one of embodiments 122 to 130, wherein the adverse event associated with administration of the first dose is a moderate allergenic adverse event or a severe allergenic adverse event.

[0360]

[0376] Embodiment 132. The method of any one of embodiments 122 to 131, wherein the first dose and the second dose are administered to the patient during a dose escalation period of oral immunotherapy.

[0361]

[0377] Embodiment 133. The method of any one of embodiments 122 to 131, wherein the first dose and the second dose are administered to the patient during a maintenance period of oral immunotherapy.

[0378] Embodiment 134. A method for adjusting the dosage of a pharmaceutical composition comprising an egg white protein during oral immunotherapy of egg allergy in a subject, the oral immunotherapy comprising (i) a dose escalation period comprising orally administering to the patient a series of increasing doses of egg white protein, and (ii) a maintenance period comprising orally administering to the patient a plurality of maintenance doses comprising egg white protein, the method comprising: orally administering to the patient a first dose of the pharmaceutical composition; orally administering to the patient a second dose of the pharmaceutical composition, wherein the second dose is reduced or skipped if the patient experiences concomitant factors associated with increased sensitivity to the allergen unrelated to administration of the first dose.

[0362]

[0379] Embodiment 135. The method of embodiment 134, wherein the cofactor associated with increased sensitivity to an allergen is an atopic disease flare, inflammation, illness, or menstruation.

[0363]

[0380] Embodiment 136. The method of embodiment 134 or 135, wherein the second dose is skipped if the patient experiences concomitant factors associated with increased sensitivity to the allergen unrelated to administration of the first dose.

[0364]

[0381] Embodiment 137. The method of embodiment 134 or 135, wherein the second dose is reduced relative to the first dose if the patient experiences concomitant factors associated with increased sensitivity to the allergen unrelated to administration of the first dose.

[0365]

[0382] Embodiment 138. The method of embodiment 137, wherein the subsequent doses of the pharmaceutical composition are reduced relative to the first dose for about one week or more before titrating the subsequent doses.

[0366]

[0383] Embodiment 139. The method of embodiment 137 or 138, wherein the subsequent doses of the pharmaceutical composition are reduced relative to the first dose for about 1 week to about 2 weeks before attempting to titrate up the subsequent doses.

[0367]

[0384] Embodiment 140. The method of any one of embodiments 137-139, wherein the subsequent doses of the pharmaceutical composition are reduced relative to the first dose for about 1 week to about 2 weeks before titrating the subsequent doses.

[0368]

[0385] Embodiment 141. The method of any one of embodiments 134 to 140, wherein the first dose and the second dose are administered to the patient during a dose escalation period of oral immunotherapy.

[0369]

[0386] Embodiment 142. The method of any one of embodiments 134 to 141, wherein the first dose and the second dose are administered to the patient during a maintenance period of oral immunotherapy.

[0370]

[0387] Embodiment 143. The method of any one of embodiments 134 to 142, wherein the cofactor associated with increased sensitivity to an allergen is unintentional exposure to a food to which the patient is allergic.

[0371]

[0388] Embodiment 144. The method of any one of embodiments 97 to 143, wherein the egg white protein in the pharmaceutical composition is a raw egg white protein.

[0372]

[0389] Embodiment 145. The method according to any one of embodiments 97 to 144, wherein the pharmaceutical composition is mixed with a food vehicle prior to administration.

[0373]

[0390] Embodiment 146. A pharmaceutical composition for use in the method according to any one of embodiments 97 to 145.

[0374]

[0391] Embodiment 147. The pharmaceutical composition according to embodiment 146, wherein the pharmaceutical composition comprises an egg white protein preparation prepared according to a method according to any one of embodiments 1 to 96.

[0375]

[0392] Embodiment 148. A pharmaceutical composition for use in the manufacture of a medicament for the method of treating egg allergy in a patient according to any one of embodiments 97 to 145.

[0376]

[0393] Embodiment 149. The pharmaceutical composition according to embodiment 148, wherein the pharmaceutical composition comprises an egg white protein preparation prepared according to a method according to any one of embodiments 1 to 96. EXAMPLES

[0377]

[0394] The present application may be better understood by reference to the following non-limiting examples, which are provided as exemplary embodiments of the present application. The following examples are presented to more fully explain the embodiments, and should not be construed in any manner as limiting the broad scope of application. Although specific embodiments of the present application are shown and described herein, it will be apparent that such embodiments are provided by way of example only. Many variations, modifications, and substitutions may occur to those skilled in the art without departing from the spirit and scope of the present invention. It should be understood that various alternatives to the embodiments described herein may be used in carrying out the methods described herein.

[0378] Example 1: Characterization of dried egg white protein powder

[0395] Lots of dried egg white protein powder were obtained from a commercial supplier and analyzed to assess the quality of the powder for use in the manufacture of egg white protein formulations. A particular characterization method involves comparing samples from one or more lots of dried egg white protein powder to a reference standard. The reference standard is a sample of dried egg white protein powder that has been monitored over time and found to have stable properties.

[0379]

[0396] Protein content in dried egg white protein powder was measured by bicinchoninic acid (BCA) assay, a total protein quantification method. Samples were prepared in water, centrifuged, and the supernatant was filtered. The supernatant was then added to a 96-well microplate, and BCA reagent was added to each well. Plates containing aliquots of samples, and several dilutions of the reference standard were developed at 37°C. Absorbance was measured at 562 nm, and sample absorbance was evaluated against a reference standard curve to measure protein concentration in the dried egg white protein powder samples. Protein content reported as a percentage by weight of protein in the dried egg white protein powder compared to the total weight of dried egg white protein is reported in Table 3.

[0380] [Table 3]

[0381]

[0397] Size exclusion chromatography (SEC-HPLC) was used as one method to establish HPLC profiles for allergenic proteins as well as an orthogonal method to measure the protein content of dried egg white protein powder. The mobile phase was a phosphate buffered saline solution (pH 6.8) that was also used to solubilize the dried egg white powder samples. The absorbance of the column elution was measured at 220 nm. The identities of the peaks were previously determined by injection of commercially purified standard products of four egg white allergenic proteins: ovalbumin (OVA), ovomucoid (OVM), ovotransferrin (OVT), and lysozyme (LYS). The SEC-HPLC chromatogram is shown in Figure 5, where the ovotransferrin, ovomucoid, ovalbumin, and lysozyme peaks are identified. The remaining peaks are not identified. Table 4 shows the peak area percent of the four egg white allergen peaks for two lots of dried egg white protein powder. Although ovomucoid and ovalbumin have different molecular weights as determined from their amino acid sequences, they co-elute using this SEC-HPLC method, which may result in similar apparent molecular weights and retention times due to protein glycosylation and protein structure.

[0382] [Table 4]

[0383]

[0398] A reversed-phase HPLC (RP-HPLC) method was developed to improve resolution and allow physical separation of egg white allergens. The RP-HPLC method was based on reversed-phase separation using a wide-bore 200 Å C4 column. A binary mobile phase gradient (mobile phase A: 0.05% TFA in water; mobile phase B: 0.05% TFA and 5% water in acetonitrile) was used to achieve baseline separation of allergenic proteins in dried egg white protein powder. Data were collected using a UV detector at a wavelength of 210 nm. Chromatographic peaks from the dried egg white protein powder sample were identified by comparing them to commercially available purified ovotransferrin, ovalbumin, ovomucoid, and lysozyme protein standards. As shown in Figure 6, RP-HPLC resolved the chromatographic peaks into ovotransferrin, ovalbumin, ovomucoid, and lysozyme. The relative amounts of ovomucoid, lysozyme, ovotransferrin, and ovalbumin (relative to total protein) in the dried egg white protein powder reference standard, as well as the relative amounts of ovomucoid, lysozyme, ovotransferrin, and ovalbumin (relative to total protein) in dried egg white protein powder lots A and B, as measured by chromatographic peak area, are shown in Table 5.

[0384] [Table 5]

[0385]

[0399] Allergenic egg white proteins were further characterized by SDS-PAGE. Proteins in the dried egg white protein powder were extracted with water, reduced using dithiothreitol, and separated using Tris-glycine gels (4-20% gradient). Coomassie blue staining was used to detect protein bands. Purified egg white protein allergens (ovomucoid, ovalbumin, lysozyme, and ovotransferrin) were obtained from commercial sources and used as comparative standards for the analysis. The SDS-PAGE data (Figure 7) confirm the presence of the four egg white protein allergen components in the analyzed samples and provide additional protein profile data to the SEC-HPLC and RP-HPLC data. In addition, the staining patterns and intensities of all bands were consistent between lots. The sample loadings for the SDS-PAGE gel shown in Figure 7 are listed in Table 6.

[0386] [Table 6]

[0387]

[0400] Immunoblot analysis of the dried egg white protein powder was also performed to further characterize the allergenic egg white proteins in the dried egg white protein powder. SDS-PAGE gels were formed as described above (see Table 6). Separated protein bands were transferred from the gel to a PVDF membrane, and the membrane was blocked with 5% skim milk blocking buffer. The membrane was then treated with pooled serum from rabbits immunized with one of ovalbumin, ovomucoid, ovotransferrin, or lysozyme. As seen in FIG. 8 for the three lots of dried egg white protein powder, the protein band distribution and intensity are consistent between lots, with bands at the expected molecular weights, and with similar immunoreactivity patterns for the egg white protein allergens.

[0388]

[0401] ELISA was used to measure the relative potency of ovomucoid in dried egg white protein powder from Lot A and Lot B against the reference standard. Proteins in dried egg white protein powder samples were extracted in phosphate buffered saline (PBS) at room temperature, vortexed, centrifuged, filtered, then serially diluted and adsorbed to ELISA plates. The plates were then blocked with 5% skim milk, washed, and incubated with primary antibody (rabbit anti-ovomucoid IgG). Sequentially, the plates were washed again and a secondary antibody conjugated to horseradish peroxidase was induced, followed by the addition of TMB substrate. The extent of anti-ovomucoid binding to ovomucoid in the samples was measured by colorimetric reaction. The data was fitted with a logistic curve fitting equation, e.g., a 4-parameter curve fit, to obtain the EC 50 The value was determined. 50 Values ​​reflect the potency of ovomucoid in the dry protein powder lot and the relative potency of the dried egg white protein powder is expressed as EC 50 The ratio of the values ​​was calculated (Table 7). Specifically, the 4-parameter curve fitting equation used was y=(AD) / (1+(X / C) B ) + D, where A is the minimum asymptote, B is the change in slope of the curve, and C is the inflection point of the curve (C is the 50% maximal effective concentration (EC 50 ) and D is the maximum asymptote. The relative potency of the sample is calculated based on the EC 50 The EC of the sample 50 The reference standard may be, for example, a previously validated lot of dried egg white protein powder or a previously validated lot of the pharmaceutical composition.

[0389] [Table 7]

[0390]

[0402] The particle size distribution of dried egg white protein powder lots was measured using laser diffraction. Dried egg white protein powder was dispersed in methanol, pumped through a flow cell, and analyzed by laser diffraction. 50 (median particle size by particle volume), D 10 (particle size at the 10th percentile of particle volume), and D 90 (particle size at 90th percentile of particle volume) was determined and is presented in Table 8.

[0391] [Table 8]

[0392]

[0403] The water activity of dried egg white protein powder was measured by placing 5 g of dried egg white protein powder in a sample cup and placing it in a water activity instrument (AquaLab 4TEV). The liquid phase and the aqueous vapor phase in the sample were equilibrated in the closed chamber of the instrument to obtain a water activity of 100% at 25° C. w , relative humidity is given. The results of this assay are shown in Table 9.

[0393] [Table 9]

[0394] Example 2: Evaluation of excipients for egg white protein formulations

[0404] During formulation and process development, five lots of egg white protein formulations were manufactured and encapsulated in a capsule dosage form. The formulations were manufactured to produce 200 mg or 300 mg doses in capsules of size 0 or 00. The contents and measured properties of each formulation are shown in Table 10. Percent deliverable mass of egg white protein formulations from a sampling of capsules.

[0395] [Table 10]

[0396]

[0405] When capsules were re-opened to deliver the contents of the dried egg white protein formulation for content uniformity testing, an average of approximately 6% to 9% by weight of the formulation remained adhered to the inside of the re-opened capsule when colloidal silicon dioxide was not included in the formulations (Lots 1-3). In addition, at-line observations noted that during encapsulation of these lots, the dried egg white protein formulation remained adhered within both ends of the re-opened capsule shell when the capsules were re-opened and shaken to simulate delivery of the powder contents.

[0397]

[0406] Colloidal silicon dioxide was subsequently added to the 300 mg dose strength blend formulation as a lubricant to improve powder flow and deliverable mass from the capsule shell during administration. The addition of colloidal silicon dioxide resulted in a significant improvement in the ease with which the powder could be delivered from the pulled apart capsule shell. The average deliverable mass ranged from 99% to 100% by weight for subsequent formulations and process development lots (lots 4-6) of the 300 mg dose strength. The delivered powder from re-opened capsules was free flowing with some soft agglomerates (from tamping during encapsulation) and broke into a powder when gently pinched between two fingers.

[0398] Example 3: Preparation of egg white protein preparation

[0407] Dried egg white protein formulations are manufactured in dosage forms with label claims of 0.2 mg, 1 mg, 6 mg, 12 mg, and 300 mg, and dried egg white protein formulations are manufactured in dosage forms with label claims of 3 mg, 20 mg, 40 mg, 80 mg, 120 mg, 160 mg, 200 mg, and 240 mg in 7 kg lots. The nominal protein content of the dried egg white protein formulations is 80% by weight, and adjustments were made for the amount of pregelatinized starch due to variations in the protein content of the dried egg white protein formulations.

[0399]

[0408] To assess the adequacy of the manufacturing process, blend uniformity was evaluated after the final mixing step of a representative lot produced at 7 kg scale. A sampler was used to take formulation samples equivalent to 1-3 capsules from the formulation produced at 10 different locations. The protein content of each sample from the set was measured using a bicinchoninic acid (BCA) assay. The results are summarized in Table 11.

[0400] [Table 11]

[0401]

[0409] Egg white protein formulations of label claims of 0.2 mg, 1 mg, 6 mg, 12 mg, and 300 mg were filled into pulled hypromellose-based capsule shells to deliver individual measured doses. Size #2 pulled capsule shells with a target powder fill weight of 180 mg were selected for dose strengths of 0.2 mg to 6 mg. Size #00 capsule shells with a target fill weight of 500 mg were selected to accommodate up to 300 mg of egg white protein with sufficient diluents, lubricants, and glidants for the higher dose strengths to allow for robust processing and deliverable mass. A Bosch GKF701 encapsulation machine was used to encapsulate the egg white protein formulations into capsules at production rates of up to 700 capsules / min. The formulations were fed into the encapsulation machine and packed into a dispensing disk using a series of tamping pins. The thickness of the dispensing disk and the position of the tamping pins can be adjusted to adjust the capsule fill weight. The empty capsule shells were opened by the encapsulation machine and a tamped slug of powder was inserted into the body of each opened capsule shell, after which the capsule shells were closed.

[0402]

[0410] The percent deliverable mass of the capsules was measured by sampling a set of 10 manufactured capsules at dose claims of 0.2 mg, 1 mg, 6 mg, 12 mg, and 300 mg. The capsules were weighed, opened, and the formulation contained therein was poured out. The mass of deliverable formulation from the capsule was measured. Compressed air was then blown into the capsule to remove any residual formulation, and the empty capsule was weighed. The percent delivered mass and RSD for each dose claim are summarized in Table 12.

[0403] [Table 12]

[0404]

[0411] The content uniformity of the capsules was also measured by comparing the protein content in the deliverable mass to the nominal dose (i.e., label claim). Protein content was measured using a BCA assay. The content uniformity of the dosage container lots at various doses is summarized in Table 13.

[0405] [Table 13]

[0406] Example 4: Oral immunotherapy trial for the treatment of egg allergy

[0412] A randomized, double-blind, placebo-controlled study of the efficacy and safety of a pharmaceutical composition containing raw dried egg white protein powder derived from chicken eggs and excipients in an oral immunotherapy regimen is conducted. A formulated placebo containing a non-active compound and corresponding excipients is used in the placebo arm of the study. A summary of the study is provided in Figure 9.

[0407]

[0413] Eligible subjects were (1) between 4 and 26 years of age, (2) had a physician-diagnosed IgE-mediated hen's egg allergy that developed within 2 hours of known oral exposure to eggs or egg-containing foods, and (3) had a ≥ 7 kU A / L, (4) develop dose-limiting allergic symptoms after ingesting a single dose of ≦300 mg dried egg white protein in a screening double-blind placebo-controlled food challenge (DBPCFC), (5) for female individuals of childbearing potential, use a reliable method of contraception, and (6) are not otherwise excluded according to the exclusion criteria.

[0408]

[0414] Exclusion criteria included: (1) history of severe or life-threatening anaphylaxis or anaphylactic shock within 60 days prior to screening; (2) history of eosinophilic esophagitis (EoE) or other eosinophilic GI disease, chronic, recurrent, or severe gastroesophageal reflux disease (GERD), symptoms of dysphagia, recurrent GI symptoms of any etiology; (3) mast cell disorder (e.g., systemic mastocytosis, urticaria pigmentosa, chronic spontaneous or chronic physical urticaria beyond simple dermatographia [e.g., cold erythema, cholinergic urticaria], or hereditary or spontaneous hemophilia). (4) having a history of uncontrolled or poorly controlled mild or moderate asthma or severe persistent asthma; (5) having a history of high-dose corticosteroid use (e.g., prednisone or equivalent, at 1 mg / kg to 2 mg / kg for more than 3 days); (6) having a history of cardiovascular disease (including uncontrolled or poorly controlled hypertension); (7) having a significant risk of becoming unstable or requiring a change in chronic treatment regimen, including malignant disease and autoimmune disease within 5 years prior to screening. (8) history of chronic disease (excluding asthma, atopic dermatitis, or allergic rhinitis) that is currently present or at risk for the disease; (9) use of beta-blockers (oral), angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, calcium channel blockers, or tricyclic antidepressants; (10) allergic reaction to 5 half-lives of the drug prior to skin prick testing (SPT) and food challenge at screening and termination and prior to the first day of dose escalation. (11) lack of an available palatable food vehicle to which the subject is not allergic; (12) hypersensitivity to wheat or oats; (13) hypersensitivity to epinephrine or any of the excipients in the epinephrine autoinjector; (14) use of any therapeutic antibody or any immunomodulatory therapy (including immunosuppressants) (except for aeroallergen or venom immunotherapy used in the maintenance period within 6 months prior to screening).(15) currently receiving or having received within 5 years prior to screening any type of egg or other food allergen immunotherapy; (16) participating in another clinical trial within 30 days or 5 half-lives of an investigational product prior to screening, whichever is longer; (17) being in the accumulation / dose escalation period of immunotherapy against any non-egg allergen; and (18) being pregnant or currently breastfeeding.

[0409]

[0415] Initial screening will include a dried egg white double-blind placebo-controlled food challenge (DBPCFC) and an open baked whole egg food challenge. The DBPCFC will be a procedure conducted under medical supervision by providing measured dried egg white and placebo, with increasing doses on day 2, by subjects, parents / caregivers, and study site staff blinded to the randomization sequence of the challenge days. Study site personnel will not be blinded to the sequence of the challenge until after completion of both days of the DBPCFC. Food challenge materials will be prepared by a designated unblinded person who will not be involved in administering, monitoring, or evaluating the results of the DBPCFC. The DBPCFC conducted in this study will follow procedures consistent with the Practical Allergy (PRACTALL) guidelines for safety, assessment, and scoring (Sampson et al., J. Allergy Clin. Immunol., vol. 130, pp. 1260-1274 (2012)). The DBPCFC procedures will be performed during screening and also at the end of the study. A summary of the screening and termination DBPCFC dosing schedule is provided in Table 14 below.

[0410] [Table 14]

[0411]

[0416] The open baked whole egg food challenge is a procedure carried out under medical supervision by feeding test food products (baked food products with eggs) at increasing measured doses. The challenge carried out under this test follows a procedure consistent with the PRACTALL guidelines for safety, evaluation, and scoring. The open baked whole egg food challenge is carried out during screening and also at the end of the study. During the challenge, subjects attempt to consume one full muffin according to the dosing schedule, with one muffin containing approximately one third of one whole egg, which corresponds to approximately 2000 mg of egg protein. The dosing schedule of the open baked whole egg food challenge at screening and at the end is provided in Table 15 below.

[0412] [Table 15]

[0413]

[0417] Eligible individuals will be randomly assigned 2:1 to blinded treatment with the pharmaceutical composition or placebo. Randomization will be stratified by baseline reactivity to baked egg in an open baked whole egg food challenge at screening. Subjects who tolerate approximately 2000 mg of cumulative baked egg protein (one muffin containing approximately one-third of one whole egg) will be allowed to consume baked egg products over the course of the study. Subjects with dose-limiting allergy symptoms during the open baked whole egg food challenge will be considered unable to tolerate baked egg and will be instructed to avoid all forms of chicken egg during the study.

[0414]

[0418] Subjects will begin an initial dose titration under medical supervision on Day 1 at the study site with stepwise dose escalation of test product (up to five single doses of 0.2 mg, 0.4 mg, 0.8 mg, 1.2 mg, and 2 mg) administered at 20-30 minute intervals as tolerated. Subjects who tolerate at least a 1.2 mg single dose on Day 1 will return to a single confirmatory 1 mg dose on Day 2. Subjects who tolerate the confirmatory 1 mg dose and do not have mild, non-dose-limiting allergic symptoms will begin the dose escalation period. Subjects who do not tolerate the 1.2 mg dose on Day 1 or do not tolerate the 1 mg dose on Day 2 will be discontinued early from the study.

[0415]

[0419] Dose escalation will be approximately 6 months (22-40 weeks) with dose escalation occurring approximately every 2 weeks. Daily doses of test product during dose escalation will be 1 mg, 3 mg, 6 mg, 12 mg, 20 mg, 40 mg, 80 mg, 120 mg, 160 mg, 200 mg, 240 mg, and 300 mg. The first dose of test product at each new dose level will be administered under medical supervision at the study site. The remaining doses at each dose level will be administered daily at home as tolerated.

[0416]

[0420] Subjects who tolerate the initial 300 mg dose, who reach a dose of 300 mg / day within 40 weeks, and who have only mild, non-dose-limiting allergic symptoms, will begin the maintenance period. Subjects who do not reach a dose of 300 mg / day within 40 weeks from Day 1 will be discontinued early from the study.

[0417]

[0421] Subjects who begin maintenance treatment will continue daily dosing with the study product at 300 mg / day for approximately 12 weeks, which may be extended up to an additional 4 weeks to accommodate dose adjustments during the final 2 weeks of maintenance. Study site visits will occur approximately every 4 weeks. Study product will be administered under medical supervision at the study site during maintenance visits. Subsequent maintenance doses, if tolerated, will be administered daily at home.

[0418]

[0422] Dose adjustments during the dose escalation or maintenance periods will be permitted in response to subjects experiencing adverse events or other co-factors, as approved by the investigator. During either the dose escalation or maintenance periods, the dose may be adjusted in response to dose-related allergic reactions according to one of the following: (1) administering the next dose of test product at the study site under medical supervision, (2) delaying dose escalation of the test product for an additional 1-2 weeks, (3) reducing the test product dose level by one or two dose levels, (4) temporarily withholding the test product, or (5) ceasing administration of the test product and discontinuing the subject from the study early. Symptom severity will induce study product dose reductions for both acute and chronic or recurrent symptoms.

[0419]

[0423] The dose may also be adjusted for reasons other than an allergic reaction caused by the pharmaceutical composition, such as asthma or other atopic disease flare-ups, illness, or menstruation. Dose reductions may range from one dose level (i.e., previous dose level) to about 50% (rounded down to the nearest feasible full dose) at the investigator's discretion. The minimum dose level is 1 mg. If the dose is reduced for reasons other than an allergic reaction to the pharmaceutical composition, the reduced dose will be given over two weeks, and the subject must be fully recovered (i.e., at baseline) for at least three days, depending on the severity of illness as assessed by the investigator, before attempting dose re-escalation at the study site. Dosing will be stopped, and the subject will be discontinued early if any of the following conditions for dose adjustment are met: at least two weeks between each titration attempt, the dose level cannot be titrated up after three consecutive failed attempts, or the dose reduction cannot be tolerated after three attempts to reduce the dose level.

[0420]

[0424] At the end of maintenance, subjects will receive a terminal DBPCFC up to a single maximum loading dose of 2000 mg dried egg white protein (4043 or 4044 mg cumulative), followed by an open baked whole egg food challenge within 7 days after the terminal DBPCFC. All subjects must tolerate a daily dose of 300 mg of test product for at least 2 consecutive weeks prior to receiving the terminal DBPCFC.

[0421]

[0425] Subjects who complete the End of Study DBPCFC and open baked whole egg food challenge will exit (complete) the study. Treatment assignment will be unblinded for subjects after study completion and after all primary data queries for the subject have been resolved. For subjects who discontinue the study early, treatment assignment will not be unblinded after study completion.

[0422]

[0426] The study endpoints tested included: (1) the proportion of subjects treated with the pharmaceutical composition who tolerate a single maximum dose of at least 300 mg raw egg white protein, at least 600 mg raw egg white protein, at least 1000 mg raw egg white protein, or at least 2000 mg raw egg white protein with only mild allergic symptoms in the end DBPCFC compared to placebo, (2) the proportion of subjects who were unable to tolerate cumulative doses of about 2000 mg baked egg protein at screening and were subsequently able to tolerate cumulative doses of about 2000 mg baked egg protein at the end of the study, and (3) treatment safety management measures.

[0423] Example 5: Oral immunotherapy trial for the treatment of egg allergy

[0427] A randomized, double-blind, placebo-controlled study of the efficacy and safety of a pharmaceutical composition containing raw dried egg white protein powder derived from chicken eggs and excipients in an oral immunotherapy regimen is conducted. A formulated placebo containing a non-active compound and corresponding excipients is used in the placebo arm of the study. A summary of the study is provided in Figure 10.

[0424]

[0428] Eligible subjects were (1) between 4 and 26 years of age, (2) had a physician-diagnosed IgE-mediated hen's egg allergy that developed within 2 hours of known oral exposure to eggs or egg-containing foods, and (3) had a ≥ 5 kU A / L, (4) develop dose-limiting allergic symptoms after ingesting a single dose of ≦300 mg dried egg white protein in a screening double-blind placebo-controlled food challenge (DBPCFC), (5) for female individuals of childbearing potential, use a reliable method of contraception, and (6) are not otherwise excluded according to the exclusion criteria.

[0425]

[0429] Exclusion criteria included: (1) history of severe or life-threatening anaphylaxis or anaphylactic shock within 60 days prior to screening; (2) history of eosinophilic esophagitis (EoE) or other eosinophilic GI disease, chronic, recurrent, or severe gastroesophageal reflux disease (GERD), symptoms of dysphagia, recurrent GI symptoms of any etiology; (3) mast cell disorder (e.g., systemic mastocytosis, urticaria pigmentosa, chronic spontaneous or chronic physical urticaria beyond simple dermatographia [e.g., cold erythema, cholinergic urticaria], or hereditary or spontaneous hemophilia). (4) having a history of uncontrolled or poorly controlled mild or moderate asthma or severe persistent asthma; (5) having a history of high-dose corticosteroid use (e.g., prednisone or equivalent, at 1 mg / kg to 2 mg / kg for more than 3 days); (6) having a history of cardiovascular disease (including uncontrolled or poorly controlled hypertension); (7) having a significant risk of becoming unstable or requiring a change in chronic treatment regimen, including malignant disease and autoimmune disease within 5 years prior to screening. (8) history of chronic disease (excluding asthma, atopic dermatitis, or allergic rhinitis) that is currently present or at risk for the disease; (9) use of beta-blockers (oral), angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, calcium channel blockers, or tricyclic antidepressants; (10) allergic reaction to 5 half-lives of the drug prior to skin prick testing (SPT) and food challenge at screening and termination and prior to the first day of dose escalation. (11) lack of an available palatable food vehicle to which the subject is not allergic; (12) hypersensitivity to wheat or oats; (13) hypersensitivity to epinephrine or any of the excipients in the epinephrine autoinjector; (14) use of any therapeutic antibody or any immunomodulatory therapy (including immunosuppressants) (except for aeroallergen or venom immunotherapy used in the maintenance period within 6 months prior to screening).(15) currently receiving or having received within 5 years prior to screening any type of egg or other food allergen immunotherapy; (16) participating in another clinical trial within 30 days or 5 half-lives of an investigational product prior to screening, whichever is longer; (17) being in the accumulation / dose escalation period of immunotherapy against any non-egg allergen; and (18) being pregnant or currently breastfeeding.

[0426]

[0430] Initial screening will include a dried egg white double-blind placebo-controlled food challenge (DBPCFC) and an open baked whole egg food challenge. The DBPCFC will be a procedure conducted under medical supervision by providing measured dried egg white and placebo, with increasing doses on day 2, by subjects, parents / caregivers, and study site staff blinded to the randomization sequence of the challenge days. Study site personnel will not be blinded to the sequence of the challenge until after completion of both days of the DBPCFC. Food challenge materials will be prepared by a designated unblinded person who will not be involved in administering, monitoring, or evaluating the results of the DBPCFC. The DBPCFC conducted in this study will follow procedures consistent with the Practical Allergy (PRACTALL) guidelines for safety, assessment, and scoring (Sampson et al., J. Allergy Clin. Immunol., vol. 130, pp. 1260-1274 (2012)). The DBPCFC procedures will be performed during screening and also at the end of the study. A summary of the screening and termination DBPCFC dosing schedule is provided in the table below.

[0427] [Table 16]

[0428]

[0431] The open baked whole egg food challenge is a procedure carried out under medical supervision by feeding test food products (baked food products with eggs) at increasing measured doses. The challenge carried out under this test follows a procedure consistent with the PRACTALL guidelines for safety, evaluation, and scoring. The open baked whole egg food challenge is carried out during screening and also at the end of the study. During the challenge, subjects attempt to consume one full muffin according to the dosing schedule, one muffin containing about one third of one whole egg, which corresponds to about 2000 mg of egg protein. The dosing schedule of the open baked whole egg food challenge at screening and at the end is provided in the following table.

[0429] [Table 17]

[0430]

[0432] Eligible individuals will be randomly assigned 2:1 to blinded treatment with the pharmaceutical composition or placebo. Randomization will be stratified by baseline reactivity to baked egg in an open baked whole egg food challenge at screening. Subjects who tolerate approximately 2000 mg of cumulative baked egg protein (one muffin containing approximately one-third of one whole egg) will be allowed to consume baked egg products over the course of the study. Subjects with dose-limiting allergy symptoms during the open baked whole egg food challenge will be considered unable to tolerate baked egg and will be instructed to avoid all forms of chicken egg during the study.

[0431]

[0433] Subjects will begin an initial dose titration under medical supervision on Day 1 at the study site with stepwise dose escalation of test product (up to five single doses of 0.2 mg, 0.4 mg, 0.8 mg, 1.0 mg, and 2 mg) administered at 20-30 minute intervals as tolerated. Subjects who tolerate at least a 1.0 mg single dose on Day 1 will return to a single confirmatory 1.0 mg dose on Day 2. Subjects who tolerate the confirmatory 1.0 mg dose and do not have mild, non-dose-limiting allergic symptoms will begin the dose escalation period. Subjects who do not tolerate a 1.0 mg dose on Day 1 or a 1 mg dose on Day 2 will be discontinued early from the study.

[0432]

[0434] Dose escalation will be approximately 6 months (22-40 weeks) with dose escalation occurring approximately every 2 weeks. Daily doses of test product during dose escalation will be 1 mg, 3 mg, 6 mg, 12 mg, 20 mg, 40 mg, 80 mg, 120 mg, 160 mg, 200 mg, 240 mg, and 300 mg. The first dose of test product at each new dose level will be administered under medical supervision at the study site. The remaining doses at each dose level will be administered daily at home as tolerated.

[0433]

[0435] Subjects who tolerate the initial 300 mg dose, who reach a dose of 300 mg / day within 40 weeks, and who have only mild, non-dose-limiting allergic symptoms, will begin the maintenance period. Subjects who do not reach a dose of 300 mg / day within 40 weeks from Day 1 will be discontinued early from the study.

[0434]

[0436] Subjects who begin maintenance treatment will continue daily dosing with the study product at 300 mg / day for approximately 12 weeks, which may be extended up to an additional 4 weeks to accommodate dose adjustments during the final 2 weeks of maintenance. Study site visits will occur approximately every 4 weeks. Study product will be administered under medical supervision at the study site during maintenance visits. Subsequent maintenance doses, if tolerated, will be administered daily at home.

[0435]

[0437] Dose adjustments during the dose escalation or maintenance periods will be permitted in response to subjects experiencing adverse events or other co-factors, as approved by the investigator. During either the dose escalation or maintenance periods, the dose may be adjusted in response to dose-related allergic reactions according to one of the following: (1) administering the next dose of test product at the study site under medical supervision, (2) delaying dose escalation of the test product for an additional 1-2 weeks, (3) reducing the test product dose level by one or two dose levels, (4) temporarily withholding the test product, or (5) ceasing administration of the test product and discontinuing the subject from the study early. Symptom severity will induce study product dose reductions for both acute and chronic or recurrent symptoms.

[0436]

[0438] The dose may also be adjusted for reasons other than an allergic reaction caused by the pharmaceutical composition, such as asthma or other atopic disease flare-ups, illness, or menstruation. Dose reductions may range from one dose level (i.e., previous dose level) to about 50% (rounded down to the nearest feasible full dose) at the investigator's discretion. The minimum dose level is 1 mg. If the dose is reduced for reasons other than an allergic reaction to the pharmaceutical composition, the reduced dose will be given over two weeks, and the subject must be fully recovered (i.e., at baseline) for at least three days, depending on the severity of illness as assessed by the investigator, before attempting dose re-escalation at the study site. Dosing will be stopped, and the subject will be discontinued early if any of the following conditions for dose adjustment are met: at least two weeks between each titration attempt, the dose level cannot be titrated up after three consecutive failed attempts, or the dose reduction cannot be tolerated after three attempts to reduce the dose level.

[0437]

[0439] At the end of maintenance, subjects will receive a terminal DBPCFC up to a single maximum loading dose of 2000 mg dried egg white protein (4043 or 4044 mg cumulative), followed by an open baked whole egg food challenge within 7 days after the terminal DBPCFC. All subjects must tolerate a daily dose of 300 mg of test product for at least 2 consecutive weeks prior to receiving the terminal DBPCFC.

[0438]

[0440] Subjects who complete the End of Study DBPCFC and open baked whole egg food challenge will exit (complete) the study. Treatment assignment will be unblinded for subjects after study completion and after all primary data queries for the subject have been resolved. For subjects who discontinue the study early, treatment assignment will not be unblinded after study completion.

[0439]

[0441] The study endpoints tested included: (1) the proportion of subjects treated with the pharmaceutical composition who tolerate a single maximum dose of at least 300 mg raw egg white protein, at least 600 mg raw egg white protein, at least 1000 mg raw egg white protein, or at least 2000 mg raw egg white protein with only mild allergic symptoms in the end DBPCFC compared to placebo, (2) the proportion of subjects who were unable to tolerate cumulative doses of about 2000 mg baked egg protein at screening and were subsequently able to tolerate cumulative doses of about 2000 mg baked egg protein at the end of the study, and (3) treatment safety management measures.

[0440] Example 6: RP-HPLC Assay

[0442] A RP-HPLC method was developed for higher resolution identification and protein profiling of dried egg white powder (drug substance) and pharmaceutical composition of egg white proteins (drug product).

[0441]

[0443] To prepare the drug substance for the assay, 50 mg of drug substance (corresponding to approximately 40 mg of egg white protein) is weighed into a 1000 ml flask and diluted with 800 ml of water. The sample is then shaken on an orbital shaker for 30 minutes, after which it is diluted with water to a concentration of 0.04 mg / ml of egg white protein. The diluted sample is then filtered through a 0.45 micron syringe filter, and the first 2 ml is discarded.

[0442]

[0444] To prepare a pharmaceutical composition (i.e., drug product) for the assay, the volume of diluent depends on the dosage strength of the sample being tested. Ten capsules from a particular dose level can be blended together and diluted with water to an egg white protein concentration of about 0.05 mg / ml. The diluted drug product sample can then be shaken on an orbital shaker for 30 minutes and diluted to a working concentration of about 0.038 mg / ml to about 0.042 mg / ml of egg white protein concentration. The sample can then be transferred to a 15 ml conical centrifuge tube and centrifuged at 3,200 rpm for 30 minutes. The clarified supernatant can then be filtered through a 0.45 micron syringe filter and the first 2 ml discarded.

[0443]

[0445] Using the improved RP-HPLC method, the major egg white protein components were fully resolved with typical retention times of approximately 15.5 min (myoglobin), 16.1 min (carbonic anhydrase), 7.8 min (ovomucoid), 12.2 min (lysozyme), 14.4 min (ovotransferrin), and 18.1 min (ovalbumin). A sample RP-HPLC chromatogram of a pharmaceutical formulation using the improved RP-HPLC method is shown in FIG. 11. Analytical samples of the drug substance or pharmaceutical product can be compared to reference standards of individual proteins (i.e., purified allergens or other egg white proteins) and / or previously validated samples of the drug substance or pharmaceutical product. The diluent of the pharmaceutical formulation showed a broad retention profile with a maximum amplitude after 22 min. The peak area of ​​each individual protein can be calculated by subtracting the area contributed by the diluent by comparison to the diluent reference standard, optionally by comparison to any other excipients only.

[0444] Example 7: Stability Study

[0446] Stability studies were performed on samples of pharmaceutical formulations of egg white protein. Samples were typically tested under accelerated and stressed stability conditions, e.g., storage conditions of 25°C to 40°C.

[0445]

[0447] In one experiment, 0.2 mg capsules were stored in high density polyethylene bottles with 1 g desiccant pouches. The bottles were stored at 5±3° C., 25° C. / 60% RH, 30° C. / 65% RH, or 40° C. / 75% RH.

[0446]

[0448] At various time points, samples were analyzed for appearance, deliverable mass, protein profile (by RP-HPLC), total protein by BCA, and relative potency by ELISA. Appearance was assessed by opening individual capsules and placing a small amount of product on a clean watch glass. The material was observed against a white background for the presence of foreign matter. Deliverable mass was assessed by weighing intact capsules, emptying the capsule into a tared container, blowing the remaining contents out of the capsule shell using compressed air, weighing the empty capsule, and then determining what percentage of the mass removed from the capsule was delivered to the tared container. Protein profile was assessed by measuring the percentage of RP-HPLC peak area contributed by Gal d1, Gal d2, Gal d3, and Gal d4. For earlier time points (1-6 months), the first RP-HPLC method described in Example 1 was used. For later time points, the improved RP-HPLC method described in Example 6 was used. Both RP-HPLC methods were used at the 6 month time point. Relative potency by ELISA and total protein by BCA were assessed as described in Example 1. BCA is reported as a percent of label claim of total egg white protein.

[0447]

[0449] The results for 0.2 mg capsules after storage at 2° C.-8° C. are summarized in the table below. At 6 months, both the older RP-HPLC method of Example 1 and the improved RP-HPLC method of Example 6 were used. The first value at 6 months corresponds to the first RP-HPLC method and the second value at 6 months corresponds to the improved RP-HPLC method.

[0448] [Table 18]

[0449]

[0450] The major allergen concentrations and relative potencies of the pharmaceutical formulations were stable for 0 to 12 months after storage at 2°C to 8°C.

[0450]

[0451] The results for 0.2 mg capsules after 25° C. / 60% RH accelerated stability storage conditions are summarized in the table below. At 6 months, the older RP-HPLC method and the improved RP-HPLC method described were used. The first value at 6 months corresponds to the first RP-HPLC method and the second value at 6 months corresponds to the improved RP-HPLC method.

[0451] [Table 19]

[0452]

[0452] Major allergen levels and relative potencies of the pharmaceutical formulations were stable for 0-12 months after storage at 25°C / 60% RH.

[0453]

[0453] Results for 0.2 mg capsules after 30°C / 65% RH accelerated stability storage conditions are summarized in the following table. At 6 months, both the older and improved RP-HPLC methods described in the previous examples were used, however, only the older RP-HPLC values ​​are reported for comparison at the T=0 time point.

[0454] [Table 20]

[0455]

[0454] Major allergen levels and relative potency of the pharmaceutical formulations were stable for 0-6 months after storage at accelerated stability conditions of 30°C / 65% RH.

[0456]

[0455] Results for 0.2 mg capsules after 40°C / 75% RH stress stability storage conditions are summarized in the following table. At the 6 month time point, both the older RP-HPLC method and the improved RP-HPLC method described in the previous example were used, however, only the older RP-HPLC method values ​​are reported for comparing the T=0, 1, and 3 month time points.

[0457] [Table 21]

[0458]

[0456] The major allergen levels and relative potency of the pharmaceutical formulation were stable for 0-6 months, even under stress-stable storage conditions of 40°C / 75% RH.

[0459]

[0457] Stability studies were completed on capsule lots containing 1 mg, 6 mg, 12 mg, and 300 mg drug doses. Results demonstrated that the pharmaceutical formulation was stable under different storage conditions across dosage levels.

[0460] Example 8: Oral immunotherapy of patients with egg allergy

[0458] A patient is treated for diagnosed egg allergy with a pharmaceutical composition of egg white protein. The patient receives an initial dose escalation, a dose escalation period, and a maintenance period according to an oral immunotherapy schedule. The indicated dose during either the dose escalation period or the maintenance period may be adjusted if the subject experiences an adverse allergic reaction to a previous dose and / or experiences concomitant factors associated with increased sensitivity to the allergen unrelated to administration of a dose of the pharmaceutical formulation.

[0461]

[0459] The patient's serum total IgE, egg white-specific IgE, total IgG4, and egg white-specific IgG4 levels can be assessed before the first administration of the pharmaceutical composition, during, and / or after oral immunotherapy. The patient's responsiveness to a dose of raw egg white and / or baked egg white can be assessed before, during, and / or after oral immunotherapy.

[0462] Example 9: Low-dose production of egg white protein preparations

[0460] A formulated pharmaceutical composition used to manufacture low doses, including 0.2 mg and 1 mg doses, for oral immunotherapy can be made as follows: The starting drug substance is dried egg white protein powder. The dried egg white protein powder is characterized to ensure consistent total protein and allergen levels, such as Gal d1, Gal d2, Gal d3, and Gal d4 levels, by comparison to a reference standard or predefined acceptance criteria. The relative potency of the egg white protein powder is confirmed, such as by ELISA against ovomucoid, and compared to predefined acceptance criteria.

[0463]

[0461] The dried egg white protein powder is mixed manually with a first portion of pregelatinized starch to form a first mixture. The first mixture is then combined and sieved through a mesh screen with a second portion of pregelatinized starch, and the combined and sieved materials are then mixed manually to form a second mixture. The second mixture is then mixed with a third portion of pregelatinized starch by a blender (e.g., a tumble blender) to form a third mixture. The third mixture is then mixed with a fourth portion of pregelatinized starch by a blender (e.g., a tumble blender) to form a fourth mixture. The fourth mixture is then mixed with a fifth portion of pregelatinized starch by a blender (e.g., a tumble blender) to form a fifth mixture. The fifth mixture is then mixed with microcrystalline cellulose by a blender (e.g., a tumble blender) and then further mixed by high shear mixing (e.g., a conical mill) to form a sixth mixture. Separately, the sixth portion of the pregelatinized starch and the magnesium stearate are mixed together to form a seventh mixture, which is then screened through a mesh screen. The seventh mixture is then mixed with the sixth mixture in a blender (e.g., a tumble blender) to form the egg white protein formulation.

[0464] Once formulated, the bulk formulation is characterized by assessment of total protein, content uniformity, individual allergen levels (such as Gal d1, Gal d2, Gal d3, and Gal d4 levels), relative potency of allergens such as by ELISA against ovomucoid, and water activity. Each assessment is judged against predefined acceptance criteria. Once the bulk formulation is validated, it is distributed into containers such as capsules or sachets. Containers from the same manufacturing batch form a lot. Individual containers, or multiple containers from the same lot, are also evaluated. The validated lot containing multiple containers is then distributed for use in oral immunotherapy of egg allergy.

[0465] Example 10: Low-dose production of egg white protein preparations

[0463] A formulated pharmaceutical composition used to manufacture low doses, including 3 mg and 6 mg doses, for oral immunotherapy can be made as follows: The starting drug substance is dried egg white protein powder. The dried egg white protein powder is characterized to ensure consistent total protein and allergen levels, such as Gal d1, Gal d2, Gal d3, and Gal d4 levels, by comparison to a reference standard or predefined acceptance criteria. The relative potency of the egg white protein powder is confirmed, such as by ELISA against ovomucoid, and compared to predefined acceptance criteria.

[0466]

[0464] The dried egg white protein powder is mixed with a first portion of pregelatinized starch to form a first mixture. The first mixture is mixed with a second portion of pregelatinized starch by high shear mixing (e.g., in a conical mill) and then further mixed in a blender (such as a tumble blender) to form a second mixture. The second mixture is then mixed with a third portion of pregelatinized starch to form a third mixture. The third mixture is then mixed with microcrystalline cellulose in a blender (e.g., a tumble blender) to form a fourth mixture and then further mixed by high shear mixing (e.g., in a conical mill). Separately, the fourth portion of pregelatinized starch and magnesium stearate are mixed together to form a fifth mixture, which is passed through a mesh screen. The sieved fifth mixture is then mixed with the fourth mixture to form the egg white protein formulation.

[0467] Once formulated, the bulk formulation is characterized by assessment of total protein, content uniformity, individual allergen levels (such as Gal d1, Gal d2, Gal d3, and Gal d4 levels), relative potency of allergens such as by ELISA against ovomucoid, and water activity. Each assessment is judged against predefined acceptance criteria. Once the bulk formulation is validated, it is distributed into containers such as capsules or sachets. Containers from the same manufacturing batch form a lot. Individual containers, or multiple containers from the same lot, are also evaluated. The validated lot containing multiple containers is then distributed for use in oral immunotherapy of egg allergy.

[0468] Example 11: Mid-dose production of egg white protein preparation

[0466] A formulated pharmaceutical composition used to manufacture an intermediate dose, including a 12 mg dose, for oral immunotherapy can be made as follows: The starting drug substance is dried egg white protein powder. The dried egg white protein powder is characterized to ensure consistent total protein and allergen levels, such as Gal d1, Gal d2, Gal d3, and Gal d4 levels, by comparison to a reference standard or predefined acceptance criteria. The relative potency of the egg white protein powder is confirmed, such as by ELISA against ovomucoid, and compared to predefined acceptance criteria.

[0469]

[0467] The dried egg white protein powder is mixed manually with a first portion of pregelatinized starch and colloidal silicon dioxide to form a first mixture. The first mixture is then mixed with a second portion of pregelatinized starch using high shear mixing (e.g., in a conical mill) to form a second mixture, and the second mixture is then further mixed in a blender (e.g., a tumble blender) at lower shear. The second mixture is then mixed with a third portion of pregelatinized starch (e.g., using a blender such as a tumble blender) to form a third mixture. The third mixture is then mixed with a fourth portion of pregelatinized starch and microcrystalline cellulose in a blender (e.g., a tumble blender) to form a fourth mixture, which is further mixed by high shear mixing (e.g., using a conical mill). Separately, a fifth portion of pregelatinized starch is mixed with magnesium stearate to form a fifth mixture, which is passed through a mesh screen. The sieved fifth mixture is then mixed with the fourth mixture to form an egg white protein preparation.

[0470] Once formulated, the bulk formulation is characterized by assessment of total protein, content uniformity, individual allergen levels (such as Gal d1, Gal d2, Gal d3, and Gal d4 levels), relative potency of allergens such as by ELISA against ovomucoid, and water activity. Each assessment is judged against predefined acceptance criteria. Once the bulk formulation is validated, it is distributed into containers such as capsules or sachets. Containers from the same manufacturing batch form a lot. Individual containers, or multiple containers from the same lot, are also evaluated. The validated lot containing multiple containers is then distributed for use in oral immunotherapy of egg allergy.

[0471] Example 12: Mid-dose production of egg protein formulation

[0469] A formulated pharmaceutical composition used to manufacture intermediate doses, including 20 mg and 40 mg doses, for oral immunotherapy can be made as follows: The starting drug substance is dried egg white protein powder. The dried egg white protein powder is characterized to ensure consistent total protein and allergen levels, such as Gal d1, Gal d2, Gal d3, and Gal d4 levels, by comparison to a reference standard or predefined acceptance criteria. The relative potency of the egg white protein powder is confirmed, such as by ELISA against ovomucoid, and compared to predefined acceptance criteria.

[0472]

[0470] The dried egg white protein powder is mixed manually with a first portion of pregelatinized starch and colloidal silicon dioxide to form a first mixture. The first mixture is then mixed with a second portion of pregelatinized starch by high shear mixing (e.g., in a conical mill) to form a second mixture, which is then further mixed in a blender (e.g., a tumble blender). The second mixture is then mixed with a third portion of pregelatinized starch and microcrystalline cellulose in a blender (such as a tumble blender), which is then further mixed by high shear mixing (e.g., in a conical mill). Separately, a fourth portion of pregelatinized starch and magnesium stearate are mixed to form a fourth mixture, which is then passed through a mesh screen. The sieved fourth mixture is then mixed with the third mixture to form the egg white protein formulation.

[0473] Once formulated, the bulk formulation is characterized by assessment of total protein, content uniformity, individual allergen levels (such as Gal d1, Gal d2, Gal d3, and Gal d4 levels), relative potency of allergens such as by ELISA against ovomucoid, and water activity. Each assessment is judged against predefined acceptance criteria. Once the bulk formulation is validated, it is distributed into containers such as capsules or sachets. Containers from the same manufacturing batch form a lot. Individual containers, or multiple containers from the same lot, are also evaluated. The validated lot containing multiple containers is then distributed for use in oral immunotherapy of egg allergy.

[0474] Example 13: High-dose production of egg white protein preparations

[0472] The formulated pharmaceutical composition used to manufacture high doses, including 120 mg, 160 mg, 200 mg, 240 mg, and 300 mg doses for oral immunotherapy, can be made as follows: Dried egg white protein powder is characterized to ensure consistent total protein and allergen levels, such as Gal d1, Gal d2, Gal d3, and Gal d4 levels, by comparison to a reference standard or predefined acceptance criteria. The relative potency of the egg white protein powder is confirmed, such as by ELISA against ovomucoid, and compared to predefined acceptance criteria.

[0475]

[0473] The dried egg white protein powder is mixed manually with a first portion of pregelatinized starch and colloidal silicon dioxide to form a first mixture. The first mixture is then mixed with microcrystalline cellulose by high shear mixing (e.g., in a conical mill) to form a second mixture, which is then further mixed in a blender (such as a tumble blender). Separately, the second portion of pregelatinized starch is mixed with magnesium stearate to form a third mixture, which is then passed through a mesh screen. The sieved third mixture is then mixed with the second mixture in a blender (such as a tumble blender) to form the egg white protein formulation.

[0476] Once formulated, the bulk formulation is characterized by assessment of total protein, content uniformity, individual allergen levels (such as Gal d1, Gal d2, Gal d3, and Gal d4 levels), relative potency of allergens such as by ELISA against ovomucoid, and water activity. Each assessment is judged against predefined acceptance criteria. Once the bulk formulation is validated, it is distributed into containers such as capsules or sachets. Containers from the same manufacturing batch form a lot. Individual containers, or multiple containers from the same lot, are also evaluated. The validated lot containing multiple containers is then distributed for use in oral immunotherapy of egg allergy. Further embodiments are as follows. [Embodiment 1] 1. A method of making an egg white protein preparation, comprising: (a) mixing dried egg white protein powder with a first amount of a first diluent to form a first mixture; (b) passing the first mixture through a mesh screen; (c) after steps (a) and (b), combining the first mixture with a second amount of the first diluent to form a second mixture; (d) mixing the second mixture with a second diluent to form a third mixture; (e) mixing the third mixture with a higher shear force than that used for mixing in step (c); (f) mixing the third mixture with a lubricant to form the egg white protein preparation. [Embodiment 2] 2. The method of embodiment 1, wherein the egg white protein preparation has about 0.05% to about 2.5% by weight of egg white protein. [Embodiment 3] 3. The method of embodiment 1 or 2, wherein the egg white protein preparation has about 0.1% to about 0.7% by weight of egg white protein. [Embodiment 4] 4. The method of any one of the preceding claims, wherein step (b) comprises passing at least a portion of the second amount of the first diluent along with the first mixture through a mesh screen. [Embodiment 5] 4. The method of any one of the preceding claims, wherein step (b) comprises passing the second amount of the first diluent with the first mixture through a mesh screen. [Embodiment 6] 6. The method of any one of the preceding claims, wherein step (c) comprises a plurality of substeps, each substep comprising: (i) adding a portion of the second amount of the first diluent to the first mixture; and (ii) mixing the portion of the second amount of the first diluent and the first mixture. [Embodiment 7] 7. The method of embodiment 6, wherein step (c) comprises three or more sub-steps. [Embodiment 8] 6. The method of any one of the preceding claims, wherein step (c) comprises continuously mixing the first mixture with the second amount of the first diluent as the second amount of the first diluent is added to the first mixture. [Embodiment 9] 9. The method of any one of the preceding claims, wherein the first mixture is mixed with the second amount of the first diluent in a tumble blender. [Embodiment 10] 10. The method of any one of the preceding claims, wherein the second mixture is mixed with the second diluent in a tumble blender. [Embodiment 11] 11. The method of any one of the preceding claims, wherein the third mixture is mixed using a conical mill. [Embodiment 12] 12. The method of any one of the preceding claims, wherein the third mixture is mixed with the lubricant in a tumble blender. [Embodiment 13] 13. The method of any one of the preceding claims, wherein a third amount of the first diluent is mixed with the third mixture. [Embodiment 14] 14. The method of embodiment 13, wherein the third amount of the first diluent and the lubricant are mixed together with the third mixture. [Embodiment 15] 15. The method of any one of the preceding claims, comprising mixing the lubricant with an additional portion of the first diluent or the second diluent prior to mixing with the third mixture. [Embodiment 16] 16. The method of embodiment 15, wherein the mixture of the lubricant and the additional portion of the first diluent or the second diluent is passed through a mesh screen before the lubricant is mixed with the third mixture. [Embodiment 17] 1. A method of making an egg white protein preparation, comprising: (a) mixing dried egg white protein powder with a first amount of a first diluent to form a first mixture; (b) combining and screening the first mixture with a second portion of the first diluent through a mesh screen to form a second mixture; (c) serially diluting the second mixture with one or more additional portions of the first diluent to form a third mixture; (d) mixing the third mixture with a second diluent to form a fourth mixture; (e) mixing an additional portion of the first diluent or the second diluent with a lubricant to form a fifth mixture; (f) mixing the fourth mixture with the fifth mixture. [Embodiment 18] 18. The method of embodiment 17, comprising mixing the second mixture prior to step (c). [Embodiment 19] 19. The method of claim 17 or 18, comprising prior to step (e) mixing the fourth mixture with a higher shear force than that used to mix the third mixture with the second diluent. [Embodiment 20] 20. The method of any one of embodiments 17-19, further comprising sieving the fifth mixture prior to step (f). [Embodiment 21] 21. The method of any one of embodiments 1-20, wherein the egg white protein preparation is substantially free of colloidal silicon dioxide. [Embodiment 22] 22. The method of any one of embodiments 1-21, wherein the egg white protein preparation is about 40% by weight to about 70% by weight of the first diluent. [Embodiment 23] 23. The method of any one of embodiments 1-22, wherein the egg white protein preparation is about 30% by weight to about 50% by weight of the second diluent. [Embodiment 24] 24. The method of any one of embodiments 1-23, wherein the egg white protein preparation has from about 0.1% by weight to about 2% by weight of the lubricant. [Embodiment 25] 1. A method of making an egg white protein preparation, comprising: (a) mixing dried egg white protein powder with a first amount of a first diluent to form a first mixture; (b) mixing a second amount of the first diluent with the first mixture to form a second mixture; (c) mixing the second mixture with a second diluent to form a third mixture; (d) mixing the third mixture with a higher shear force than that used for mixing in step (b); (e) mixing the third mixture with a lubricant to form the egg white protein preparation. [Embodiment 26] 26. The method of embodiment 25, wherein the dried egg white protein powder is mixed with the first amount of the first diluent in step (a) at a higher shear force than that used for mixing in step (b). [Embodiment 27] 27. The method of embodiment 25 or 26, wherein the egg white protein preparation has about 1% to about 70% by weight of egg white protein. [Embodiment 28] 28. The method of any one of embodiments 25-27, wherein the dried egg white protein powder is mixed with the first amount of the first diluent in step (a) using a conical mill. [Embodiment 29] 29. The method according to any one of embodiments 25 to 28, wherein step (b) comprises two mixing sub-steps, one mixing sub-step being at a higher shear force than the other mixing sub-step. [Embodiment 30] 30. The method of any one of claims 25-29, wherein step (c) further comprises mixing the second mixture with an additional amount of the first diluent prior to mixing with the second diluent to form the third mixture. [Embodiment 31] 30. The method of any one of embodiments 25-29, wherein step (c) further comprises combining the second mixture with additional amounts of the first diluent and the second diluent to form the third mixture. [Embodiment 32] 32. The method of any one of embodiments 25-31, wherein the third mixture is mixed in step (d) using a conical mill. [Embodiment 33] 33. The method of any one of embodiments 25-32, wherein the second amount of the first diluent and the first mixture are mixed in a tumble blender. [Embodiment 34] 34. The method of any one of embodiments 25-33, wherein the second mixture is mixed with the second diluent in a tumble blender. [Embodiment 35] 34. The method of any one of embodiments 25-33, wherein a third amount of the first diluent is mixed with the third mixture. [Embodiment 36] 36. The method of embodiment 35, wherein the third amount of the first diluent and the lubricant are mixed together with the third mixture. [Embodiment 37] 37. The method of any one of embodiments 25-36, wherein step (e) comprises: (i) mixing a portion of the third mixture with a lubricant; (ii) passing the mixture of (i) through a mesh screen; and (iii) mixing the mixture of (ii) with an additional portion of the third mixture to form the egg white protein preparation. [Embodiment 38] 38. The method of any one of embodiments 25-37, wherein the third mixture is mixed with the lubricant in a tumble blender. [Embodiment 39] 39. The method of any one of embodiments 25-38, wherein the egg white protein preparation is about 9% to about 85% by weight of the first diluent. [Embodiment 40] 40. The method of any one of embodiments 25-39, wherein the egg white protein preparation is about 10% by weight to about 50% by weight of the second diluent. [Embodiment 41] The method of any one of embodiments 25 to 40, wherein the egg white protein preparation is about 10% by weight to about 20% by weight of the second diluent. [Embodiment 42] 42. The method of any one of embodiments 25-41, wherein the egg white protein preparation has from about 0.1% by weight to about 2% by weight of the lubricant. [Embodiment 43] 43. The method of any one of embodiments 25-42, wherein the egg white protein preparation comprises a lubricant. [Embodiment 44] 44. The method of embodiment 43, wherein the lubricant is mixed with the egg white protein powder and the first amount of the first diluent during or prior to step (a). [Embodiment 45] The method of embodiment 43 or 44, wherein the lubricant is colloidal silicon dioxide. [Embodiment 46] 46. ​​The method of any one of embodiments 25-45, wherein the egg white protein preparation is about 2% to about 70% by weight of egg white protein. [Embodiment 47] 45. The method of any one of embodiments 25-44, wherein the egg white protein preparation is substantially free of colloidal silicon dioxide. [Embodiment 48] 48. The method of embodiment 47, wherein the egg white protein preparation is about 1% to about 5% by weight egg white protein. [Embodiment 49] 1. A method of making an egg white protein preparation, comprising: (a) mixing dried egg white protein powder with a first diluent and a lubricant to form a first mixture; (b) mixing a second diluent and the first mixture under a higher shear force than that used for mixing in step (a) to form a second mixture; (c) mixing the second mixture and a lubricant to form the egg white protein preparation. [Embodiment 50] 50. The method of embodiment 49, wherein the egg white protein preparation has about 50% to about 80% by weight of egg white protein. [Embodiment 51] 51. The method of embodiment 49 or 50, wherein the dried egg white protein powder, the first diluent, and the lubricant are mixed in a tumble blender. [Embodiment 52] 52. The method of any one of embodiments 49-51, wherein the second diluent and the first mixture are mixed in step (b) using a conical mill. [Embodiment 53] 53. The method of any one of embodiments 49-52, wherein the second mixture and the lubricant are mixed in a tumble blender. [Embodiment 54] 54. The method of any one of embodiments 49-53, wherein a second amount of the first diluent is mixed with the second mixture. [Embodiment 55] 55. The method of embodiment 54, wherein the second amount of the first diluent and the lubricant are combined and mixed with the second mixture. [Embodiment 56] 56. The method of any one of embodiments 49-55, wherein the lubricant comprises colloidal silicon dioxide. [Embodiment 57] 57. The method of any one of embodiments 49-56, wherein step (c) comprises (i) mixing a portion of the second mixture with the lubricant; (ii) passing the portion of the second mixture and the lubricant through a mesh screen; and (iii) mixing (ii) with an additional portion of the second mixture to form the egg white protein preparation. [Embodiment 58] 58. The method of any one of embodiments 1-57, wherein the egg white protein preparation is produced in a lot size of about 5 kg or more. [Embodiment 59] 59. The method of embodiment 58, wherein the egg white protein preparation is produced in a lot size of about 5 kg to about 50 kg. [Embodiment 60] 60. The method of any one of embodiments 1-59, comprising measuring the egg white protein blend uniformity of the egg white protein preparation. [Embodiment 61] 61. The method of any one of embodiments 1-60, wherein the egg white protein preparation has an egg white protein blend uniformity relative standard deviation (RSD) of about 15% or less. [Embodiment 62] 62. The method of any one of embodiments 1-61, comprising packaging the egg white protein preparation in a multi-dose container. [Embodiment 63] 63. The method of embodiment 62, wherein the dosage container is a capsule or sachet. [Embodiment 64] 64. The method of embodiment 62 or 63, comprising measuring the egg white protein content uniformity of the plurality of dosage containers. [Embodiment 65] 65. The method of any one of embodiments 62-64, wherein the plurality of dose containers has an egg white protein content uniformity relative standard deviation (RDS) of about 15% or less. [Embodiment 66] 66. The method of any one of the preceding embodiments, wherein the first diluent is pregelatinized starch. [Embodiment 67] 67. The method of any one of the preceding embodiments, wherein the second diluent is microcrystalline cellulose. [Embodiment 68] 68. The method of any one of the preceding embodiments, wherein the lubricant is magnesium stearate. [Embodiment 69] 69. The method of any one of the preceding embodiments, wherein the dried egg white protein powder comprises about 50% to about 90% by weight of egg white protein. [Embodiment 70] 70. The method of any one of the preceding embodiments, wherein forming the dried egg white protein powder comprises spray drying liquid egg white. [Embodiment 71] 71. The method of any one of the preceding claims, wherein the egg white protein powder is deglucose depleted. [Embodiment 72] 72. The method of any one of the preceding embodiments, wherein the dried egg white powder is pasteurized. [Embodiment 73] 73. The method of any one of embodiments 1-72, wherein the dried egg white protein powder is derived from chicken eggs. [Embodiment 74] 74. The method of any one of the preceding embodiments, further comprising characterizing ovomucoid, ovalbumin, ovotransferrin, or lysozyme in the dried egg white protein powder. [Embodiment 75] 75. The method of any one of embodiments 1-74, further comprising characterizing ovomucoid, ovalbumin, ovotransferrin, or lysozyme in the egg white protein preparation. [Embodiment 76] 76. The method of embodiment 74 or 75, wherein characterizing ovomucoid, ovalbumin, ovotransferrin, or lysozyme comprises obtaining a high performance liquid chromatography (HPLC) profile. [Embodiment 77] 77. The method of embodiment 76, wherein said HPLC profile is a reverse phase HPLC (RP-HPLC) profile. [Embodiment 78] 77. The method of embodiment 76, wherein said HPLC profile is a size exclusion chromatography HPLC (SEC-HPLC) profile. [Embodiment 79] The method of any one of embodiments 76 to 78, comprising comparing the obtained HPLC profile with a reference HPLC profile. [Embodiment 80] 80. The method of any one of embodiments 74-79, wherein characterizing the ovomucoid, ovalbumin, ovotransferrin, or lysozyme comprises quantifying the amount of ovomucoid, ovalbumin, ovotransferrin, or lysozyme. [Embodiment 81] 81. The method of embodiment 80, wherein quantifying the amount of ovomucoid, ovalbumin, ovotransferrin, or lysozyme comprises measuring the amount of ovomucoid, ovalbumin, ovotransferrin, or lysozyme relative to total protein in the egg white protein powder or egg white protein preparation. [Embodiment 82] The method of embodiment 80, wherein quantifying the amount of ovomucoid, ovalbumin, ovotransferrin, or lysozyme comprises measuring the amount of ovomucoid, ovalbumin, ovotransferrin, or lysozyme relative to the total amount of ovomucoid, ovalbumin, ovotransferrin, and lysozyme in the egg white protein powder or egg white protein formulation. [Embodiment 83] 83. The method of any one of embodiments 74-82, wherein characterizing ovomucoid, ovalbumin, ovotransferrin, or lysozyme comprises measuring potency of ovomucoid, ovalbumin, ovotransferrin, or lysozyme in the egg white protein powder or egg white protein formulation. [Embodiment 84] The method of embodiment 83, wherein the potency of ovomucoid, ovalbumin, ovotransferrin, or lysozyme in the egg white protein powder or egg white protein preparation is measured relative to the potency of ovomucoid, ovalbumin, ovotransferrin, or lysozyme in a reference sample. [Embodiment 85] The method of embodiment 83 or 84, wherein said potency of ovomucoid, ovalbumin, ovotransferrin, or lysozyme is measured using an immunoassay. [Embodiment 86] 86. The method of embodiment 85, wherein said im...

Claims

1. 1. A method of making an egg white protein preparation, comprising: (I) (a) mixing a dry egg white protein powder consisting solely of 10-20% ovomucoid, 0.1-3% lysozyme, 1-5% ovotransferrin, and 70-80% ovalbumin by total weight of egg white protein with a first amount of pregelatinized starch to form a first mixture; (b) passing the first mixture through a mesh screen; (c) mixing the first mixture with a second amount of the pregelatinized starch at a higher shear force than step (a) to form a second mixture; (d) mixing the second mixture with a third portion of pregelatinized starch to form a third mixture; (e) mixing the third mixture with a fourth portion of pregelatinized starch and microcrystalline cellulose at a shear force higher than that used for mixing in step (d) to form a fourth mixture; (f) separately combining a fifth portion of the pregelatinized starch with magnesium stearate to form a fifth mixture; (g) passing the fifth mixture through a mesh screen; (h) mixing the fourth mixture with the fifth mixture to form the egg white protein preparation; wherein the egg white protein preparation consists solely of egg white proteins ovomucoid, lysozyme, ovotransferrin, and ovalbumin, pregelatinized starch, microcrystalline cellulose, and magnesium stearate. (II) (a) mixing a dry egg white protein powder consisting solely of 10-20% ovomucoid, 0.1-3% lysozyme, 1-5% ovotransferrin, and 70-80% ovalbumin by total weight of egg white protein with a first amount of pregelatinized starch to form a first mixture; (b) combining and sieving the first mixture with a second portion of the pregelatinized starch through a mesh screen to form a second mixture; (c) serially diluting the second mixture 1 to 3 times with one or more additional portions of the pregelatinized starch to form a third mixture; (d) mixing the third mixture with microcrystalline cellulose at a shear force higher than that used for mixing in step (c) to form a fourth mixture; (e) separately mixing an additional portion of the pregelatinized starch with magnesium stearate to form a fifth mixture; (f) mixing the fourth mixture with the fifth mixture to form the egg white protein preparation; wherein the egg white protein preparation consists solely of egg white proteins ovomucoid, lysozyme, ovotransferrin, and ovalbumin, pregelatinized starch, microcrystalline cellulose, and magnesium stearate; or (III) (a) mixing a dry egg white protein powder consisting solely of 10-20% ovomucoid, 0.1-3% lysozyme, 1-5% ovotransferrin, and 70-80% ovalbumin by total weight of egg white protein with a first amount of pregelatinized starch to form a first mixture; (b) mixing a second amount of the pregelatinized starch with the first mixture at a higher shear force than that used for mixing in step (a) to form a second mixture; (c) mixing the second mixture with a third portion of pregelatinized starch to form a third mixture; (d) mixing the third mixture with microcrystalline cellulose at a shear force higher than that used for mixing in step (a) to form a fourth mixture; (e) separately blending a fourth portion of the pregelatinized starch with the magnesium stearate to form a fifth blend and passing through a mesh screen; (f) mixing the fourth mixture with the fifth mixture to form the egg white protein preparation, wherein the egg white protein preparation consists solely of egg white proteins consisting of ovomucoid, lysozyme, ovotransferrin, and ovalbumin, pregelatinized starch, microcrystalline cellulose, and magnesium stearate.

2. (I) comprising steps (a) to (h), 2. The method of claim 1, wherein the egg white protein preparation has 0.05% to 2.5% by weight of egg white protein, or 0.1% to 0.7% by weight of egg white protein.

3. (I) comprising steps (a) to (h), step (b) comprises passing at least a portion of the second amount of pregelatinized starch with the first mixture through a mesh screen; and 3. The method of claim 1 or 2, wherein step (c) comprises continuously mixing the first mixture with the second amount of pregelatinized starch as the second amount of pregelatinized starch is added to the first mixture.

4. The method according to any one of claims 1 to 3, wherein step (a) of (I) is mixed by hand, steps (d), (f), (g) and (h) of (I) are mixed by a tumble blender, and steps (c) and (e) of (I) are mixed by a tumble blender and then further mixed by a conical mill.

5. 5. The method according to any one of claims 1 to 4, wherein the egg white protein preparation has from 40% to 70% by weight of the pregelatinized starch.

6. 6. The method of any one of claims 1 to 5, wherein the egg white protein preparation has 30% to 50% by weight of the microcrystalline cellulose.

7. 7. The method of any one of claims 1 to 6, wherein the egg white protein preparation has from 0.1% to 2% by weight of the magnesium stearate.

8. 2. The method of claim 1, comprising steps (a) to (f) of (III), wherein the egg white protein preparation has 1% to 70% by weight of egg white protein.

9. (III) comprising steps (a) to (f), step (b) comprises two mixing sub-steps, one mixing sub-step being at a higher shear force than the other mixing sub-step; 9. The method of claim 1 or 8, wherein step (c) further comprises mixing the second mixture with an additional amount of the pregelatinized starch prior to mixing with the microcrystalline cellulose to form the third mixture; or wherein step (c) further comprises mixing the second mixture together with additional amounts of the pregelatinized starch and the microcrystalline cellulose to form the third mixture.

10. (III) comprising steps (a) to (f), 10. The method according to any one of claims 1, 8 and 9, wherein the egg white protein preparation has from 9% to 85% by weight of the pregelatinized starch.

11. (III) comprising steps (a) to (f), 11. The method of any one of claims 1 and 8-10, wherein the egg white protein preparation has 10% to 50% by weight of the microcrystalline cellulose or 10% to 20% by weight of the microcrystalline cellulose.

12. (III) comprising steps (a) to (f), 12. The method of any one of claims 1 and 8-11, wherein the egg white protein preparation has from 0.1% to 2% by weight of the magnesium stearate.

13. (III) comprising steps (a) to (f), 13. The method according to any one of claims 1 and 8 to 12, wherein the egg white protein preparation has from 2% to 70% by weight of egg white protein.

14. The method according to claim 14, comprising steps (a) to (f) of (III), 2. The method of claim 1, wherein the egg white protein preparation has 1% to 5% egg white protein by weight.

15. measuring the egg white protein blend uniformity of the egg white protein formulation; 15. The method of any one of the preceding claims, wherein the egg white protein blend uniformity relative standard deviation (RSD) is less than or equal to 15%.

16. 16. The method of any one of claims 1 to 15, further comprising characterizing ovomucoid, ovalbumin, ovotransferrin and lysozyme in the dried egg white protein powder.

17. An egg white protein preparation consisting only of dried egg white protein powder which consists only of 10-20% ovomucoid, 0.1-3% lysozyme, 1-5% ovotransferrin, and 70-80% ovalbumin based on the total mass of egg white protein, pregelatinized starch, microcrystalline cellulose, and magnesium stearate.

18. 18. The egg white protein preparation of claim 17, wherein the egg white protein preparation comprises 0.1% to 3.5% egg white protein by weight.

19. 1. A pharmaceutical composition comprising egg white protein for use in a method for treating egg allergy in a patient, comprising: the method comprising orally administering to a patient multiple doses of the pharmaceutical composition consisting solely of egg white protein, pregelatinized starch, microcrystalline cellulose, and magnesium stearate, the pharmaceutical composition consisting solely of 10-20% ovomucoid, 0.1-3% lysozyme, 1-5% ovotransferrin, and 70-80% ovalbumin by total weight of egg white protein, according to an oral immunotherapy schedule; The oral immunotherapy schedule comprises: (a) a dose escalation period comprising orally administering to the patient a series of ascending doses of egg white protein ranging from 1 mg to 300 mg, wherein a given dose is administered to the patient for at least two weeks before the dose is escalated, and wherein the dose escalation period is 20 weeks to 44 weeks in length; (b) a maintenance period comprising orally administering to the patient a number of maintenance doses comprising egg white protein, wherein the maintenance period is 12 weeks or greater in length.

20. The patient's serum level of egg white specific IgE (ew-IgE) was 7 kU at the start of the treatment. Ａ 20. The pharmaceutical composition of claim 19, wherein the total amount of the compound is 100 mM MgCl2 or more.

21. 21. The pharmaceutical composition of claim 19 or 20, wherein the series of ascending doses administered to the patient during the dose escalation period comprises at least a 1 mg dose of egg white protein, and at least a 300 mg dose of egg white protein.

22. 22. The pharmaceutical composition of any one of claims 19-21, wherein the multiple maintenance doses administered to the patient during the maintenance period comprise 300 mg egg white protein.

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