Formulation for egg oral immunotherapy, preparation method and treatment for egg allergy

文档序号：23284 发布日期：2021-09-21 浏览：34次中文

阅读说明：本技术 用于卵口服免疫疗法的制剂、制备方法和用于卵变态反应的治疗 (Formulation for egg oral immunotherapy, preparation method and treatment for egg allergy ) 是由 R·J·西蒙 N·W·伯林格 M·桑德 C·卡普勒 K·R·布朗 S·G·迪利 D·阿德于 2019-12-17 设计创作，主要内容包括：本文描述了卵清蛋白质制剂、含有卵清蛋白质制剂的剂量容器、制备卵清蛋白质制剂和剂量容器的方法、以及控制卵清蛋白质制剂的质量的方法、用于制备卵清蛋白质制剂的材料(诸如干燥卵清蛋白质粉末)以及制备剂量容器的材料。本文还描述了用于治疗卵变态反应的口服免疫疗法方法。该方法包括根据口服免疫疗法计划表向患者口服施用一定剂量的包含卵清蛋白质的药物组合物。本文还描述了如果患者经历与剂量施用有关的不良事件或与对与剂量施用无关的变应原增加的敏感性相关联的并发因素则在口服免疫疗法期间调整药物组合物的剂量的方法。(Described herein are egg white protein formulations, dosage containers containing egg white protein formulations, methods of making egg white protein formulations and dosage containers, and methods of controlling the quality of egg white protein formulations, materials for making egg white protein formulations (such as dried egg white protein powder), and materials for making dosage containers. Also described herein are oral immunotherapy methods for treating egg allergy. The method comprises orally administering to the patient a dose of a pharmaceutical composition comprising an egg white protein according to an oral immunotherapy schedule. Also described herein are methods of adjusting the dose of a pharmaceutical composition during oral immunotherapy if a patient experiences an adverse event related to dose administration or a complication associated with increased sensitivity to an allergen not related to dose administration.)

1. A method of preparing an egg white protein formulation, the method comprising:

(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 at a higher shear force than the shear force used for mixing in step (c); and

(f) Mixing the third mixture with a lubricant to form the egg white protein formulation.

2. The method of claim 1, wherein the egg white protein formulation has about 0.05% to about 2.5% egg white protein by weight.

3. The method of claim 1 or 2, wherein the egg white protein formulation has about 0.1% to about 0.7% egg white protein by weight.

4. The method of any one of claims 1 to 3, wherein step (b) comprises passing at least part of the second amount of the first diluent and the first mixture through the mesh screen.

5. The method of any one of claims 1 to 3, wherein step (b) comprises passing the second amount of the first diluent and the first mixture through the mesh screen.

6. The method according to any one of claims 1 to 5, wherein step (c) comprises a plurality of sub-steps, wherein each sub-step comprises (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 with the first mixture.

7. The method of claim 6, wherein step (c) comprises three or more substeps.

8. The method of any one of claims 1 to 5, wherein step (c) comprises continuously mixing the first mixture and the second amount of the first diluent while the second amount of the first diluent is added to the first mixture.

9. The method of any one of claims 1 to 8, wherein the first mixture and the second amount of the first diluent are mixed in a drum blender.

10. The method of any one of claims 1 to 9, wherein the second mixture is mixed with the second diluent in a drum blender.

11. The method of any one of claims 1 to 10, wherein the third mixture is mixed using a cone mill.

12. The method of any one of claims 1 to 11, wherein the third mixture is mixed with the lubricant in a drum blender.

13. The method of any one of claims 1 to 12, wherein a third amount of the first diluent is mixed with the third mixture.

14. The method of claim 13, wherein the third amount of the first diluent and the lubricant are co-mixed with the third mixture.

15. The method of any one of claims 1 to 14, comprising mixing the lubricant with a further portion of the first diluent or the second diluent prior to mixing the lubricant with the third mixture.

16. The method of claim 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 prior to mixing the lubricant with the third mixture.

17. A method of preparing an egg white protein formulation, the method comprising:

(a) mixing a dry egg white protein powder with a first amount of a first diluent to form a first mixture;

(b) co-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; and

(f) Mixing the fourth mixture with the fifth mixture.

18. The method of claim 17, comprising mixing the second mixture prior to step (c).

19. The method of claim 17 or 18, comprising mixing the fourth mixture prior to step (e) using a higher shear force than the shear force used to mix the third mixture with the second diluent.

20. The method of any one of claims 17 to 19, comprising sieving the fifth mixture prior to step (f).

21. The method of any one of claims 1-20, wherein the egg white protein preparation is substantially free of colloidal silica.

22. The method of any one of claims 1-21, wherein the egg white protein formulation has from about 40% to about 70% by weight of the first diluent.

23. The method of any one of claims 1-22, wherein the egg white protein formulation has from about 30% to about 50% by weight of the second diluent.

24. The method of any one of claims 1-23, wherein the egg white protein formulation has from about 0.1% to about 2% by weight of the lubricant.

25. A method of preparing an egg white protein formulation, the method comprising:

(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;

(d) mixing the third mixture at a higher shear force than the shear force used for mixing in step (b); and

(e) mixing the third mixture with a lubricant to form the egg white protein formulation.

26. The method of claim 25, wherein dry egg white protein powder is mixed with the first amount of the first diluent in step (a) at a higher shear force than the shear force used for mixing in step (b).

27. The method of claim 25 or 26, wherein the egg white protein formulation has about 1% to about 70% egg white protein by weight.

28. The method of any one of claims 25-27, wherein the dried egg white protein powder is mixed with the first amount of the first diluent in step (a) using a cone mill.

29. A method according to any one of claims 25 to 28, wherein step (b) comprises two mixing sub-steps, wherein one mixing sub-step is at a higher shear force than the other mixing sub-step.

30. The method of any one of claims 25 to 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.

31. The method of any one of claims 25 to 29, wherein step (c) further comprises co-mixing the second mixture with additional amounts of the first diluent and the second diluent to form the third mixture.

32. The process of any one of claims 25 to 31, mixing the third mixture in step (d) using a cone mill.

33. The method of any one of claims 25 to 32, wherein the second amount of the first diluent and the first mixture are mixed in a drum blender.

34. The method of any one of claims 25 to 33, wherein the second mixture is mixed with the second diluent in a drum blender.

35. The method of any one of claims 25 to 33, wherein a third amount of the first diluent is mixed with the third mixture.

36. The method of claim 35, wherein the third amount of the first diluent and the lubricant are co-mixed with the third mixture.

37. The method of any one of claims 25 to 36, wherein step (e) comprises: (i) mixing a portion of the third mixture with a lubricant; (ii) (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.

38. The method of any one of claims 25 to 37, wherein the third mixture is mixed with the lubricant in a drum blender.

39. The method of any one of claims 25-38, wherein the egg white protein formulation has about 9% to about 85% by weight of the first diluent.

40. The method of any one of claims 25-39, wherein the egg white protein formulation has from about 10% to about 50% by weight of the second diluent.

41. The method of any one of claims 25-40, wherein the egg white protein formulation has from about 10% to about 20% by weight of the second diluent.

42. The method of any one of claims 25-41, wherein the egg white protein formulation has from about 0.1% to about 2% by weight of the lubricant.

43. The method of any one of claims 25-42, wherein the egg white protein formulation comprises a glidant.

44. The method of claim 43, wherein the glidant is mixed with the egg white protein powder and the first amount of the first diluent during or prior to step (a).

45. The method of claim 43 or 44, wherein the glidant is colloidal silicon dioxide.

46. The method of any one of claims 25-45, wherein the egg white protein formulation has about 2% to about 70% egg white protein by weight.

47. The method of any one of claims 25-44, wherein the egg white protein preparation is substantially free of colloidal silica.

48. The method of claim 47, wherein the egg white protein preparation has about 1% to about 5% egg white protein by weight.

49. A method of preparing an egg white protein formulation, the method comprising:

(a) mixing a dry egg white protein powder, a first diluent, and a glidant to form a first mixture;

(b) mixing a second diluent with the first mixture at a higher shear force than the shear force used for mixing in step (a) to form a second mixture;

50. The method of claim 49, wherein the egg white protein preparation has about 50% to about 80% egg white protein by weight.

51. The method of claim 49 or 50, wherein the dried egg white protein powder, the first diluent, and the glidant are mixed in a drum blender.

52. The method of any one of claims 49 to 51, wherein the second diluent and the first mixture are mixed in step (b) using a cone mill.

53. The method of any one of claims 49-52, wherein the second mixture and the lubricant are mixed in a drum blender.

54. The method of any one of claims 49 to 53, wherein a second amount of the first diluent is mixed with the second mixture.

55. The method of claim 54, wherein the second amount of the first diluent and the lubricant are co-mixed with the second mixture.

56. The method of any one of claims 49-55, wherein the glidant comprises colloidal silicon dioxide.

57. The method of any one of claims 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.

58. The method of any one of claims 1-57, wherein the egg white protein preparation is prepared in a batch size of about 5kg or greater.

59. The method of claim 58, wherein the egg white protein preparation is prepared in a batch size of from about 5kg to about 50 kg.

60. The method of any one of claims 1 to 59, comprising determining an egg white protein blend uniformity of the egg white protein formulation.

61. The method of any one of claims 1-60, wherein the egg white protein formulation has an egg white protein blend uniformity Relative Standard Deviation (RSD) of about 15% or less.

62. The method of any one of claims 1 to 61, comprising packaging the egg white protein formulation in a plurality of dosage containers.

63. The method of claim 62, wherein the dose container is a capsule or a pouch.

64. The method of claim 62 or 63, comprising determining ovalbumin content uniformity for the plurality of dosage containers.

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

66. The method of any one of claims 1 to 65, wherein the first diluent is pregelatinized starch.

67. The method of any one of claims 1 to 66, wherein the second diluent is microcrystalline cellulose.

68. The method of any one of claims 1-67, wherein the lubricant is magnesium stearate.

69. The method of any one of claims 1-68, wherein the dried egg white protein powder comprises about 50% to about 90% by weight egg white protein.

70. The method of any one of claims 1-69, wherein the forming of the dried egg white protein powder comprises spray drying liquid egg white.

71. The method of any one of claims 1-70, wherein the egg white protein powder has been depleted of glucose.

72. The method of any one of claims 1-71, wherein the dried egg white protein powder has been pasteurized.

73. The method of any one of claims 1-72, wherein the dried egg white protein powder is derived from an egg.

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

75. The method of any one of claims 1 to 74, further comprising characterizing ovomucoid, ovalbumin, ovotransferrin, or lysozyme in the egg white protein formulation.

76. The method of claim 74 or 75, wherein characterizing ovomucoid, ovalbumin, ovotransferrin, or lysozyme comprises obtaining a High Performance Liquid Chromatography (HPLC) profile.

77. The method of claim 76, wherein the HPLC profile is a reverse phase HPLC (RP-HPLC) profile.

78. The method of claim 76, wherein the HPLC profile is a size exclusion chromatography HPLC (SEC-HPLC) profile.

79. The method of any one of claims 76 to 78, comprising comparing the obtained HPLC profile with a reference HPLC profile.

80. The method of any one of claims 74-79, wherein characterizing ovomucoid, ovalbumin, ovotransferrin, or lysozyme comprises quantifying the amount of ovomucoid, ovalbumin, ovotransferrin, or lysozyme.

81. The method of claim 80, wherein quantifying the amount of ovomucoid, ovalbumin, ovotransferrin, or lysozyme comprises measuring the amount of ovomucoid, ovalbumin, ovotransferrin, or lysozyme compared to the total protein in the egg white protein powder or the egg white protein preparation.

82. The method of claim 80, wherein quantifying the amount of ovomucoid, ovalbumin, ovotransferrin, or lysozyme comprises measuring the amount of ovomucoid, ovalbumin, ovotransferrin, or lysozyme compared to the total amount of ovomucoid, ovalbumin, ovotransferrin, or lysozyme in the egg white protein powder or the egg white protein formulation.

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

84. The method of claim 83, wherein the potency of ovomucoid, ovalbumin, ovotransferrin, or lysozyme in the egg white protein powder or the egg white protein formulation is measured relative to the potency of ovomucoid, ovalbumin, ovotransferrin, or lysozyme in a reference sample.

85. The method of claim 83 or 84, wherein the potency of ovomucoid, ovalbumin, ovotransferrin, or lysozyme is measured using an immunoassay.

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

87. The method of claim 85, wherein the immunoassay comprises the use of an antibody library 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.

88. The method of claims 86 and 87, wherein the antibody is an IgE antibody or an IgG antibody.

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

90. An egg white protein preparation made according to the method of any one of claims 1 to 89.

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.

92. An egg white protein preparation according to claim 91 wherein the egg white protein preparation comprises about 0.1% to about 3.5% by weight egg white protein.

93. An egg white protein formulation as in claim 91 or 92 wherein the first diluent is pregelatinized starch.

94. An egg white protein formulation as in any one of claims 91 to 93 wherein the second diluent is microcrystalline cellulose.

95. An egg white protein formulation according to any one of claims 91 to 94, wherein the lubricant is magnesium stearate.

96. The egg white protein formulation of any one of claims 91 to 95, wherein the egg white protein formulation consists essentially of the egg white protein powder, the first diluent, the second diluent, and the lubricant.

97. A method of treating egg allergy in a patient, comprising:

orally administering to the patient a plurality of doses of a pharmaceutical composition comprising an egg white protein according to an oral immunotherapy schedule comprising:

(a) an up-dosing phase comprising orally administering to the patient a series of ascending doses of egg white protein of about 1mg to about 300mg, wherein a given dose is administered to the patient for at least two weeks prior to the dose escalation, and wherein the length of the up-dosing phase is from about 20 weeks to about 44 weeks; and

(b) a maintenance phase comprising orally administering to the patient a plurality of maintenance doses comprising egg white protein, wherein the maintenance phase is about 12 weeks or more in length.

98. The method of claim 97, wherein the patient has about 7kU at the start of treatment_A(ii) egg white-specific serum IgE (ew-IgE) levels of/L or higher.

99. The method of claim 97, wherein the patient has about 5kU at the start of treatment_A(ii) egg white-specific serum IgE (ew-IgE) levels of/L or higher.

100. The method of any one of claims 97-99, wherein the series of escalating doses administered to the patient during the up-dosing phase comprises at least a 1mg dose of egg white protein and a 300mg dose of egg white protein.

101. The method of any one of claims 97-100, wherein the series of escalating doses administered to the patient during the upregulating dose phase comprises at least 10 different doses of egg white protein.

102. The method of any one of claims 97-101, wherein the series of escalating doses administered to the patient during the up-dosing phase comprises doses of about 1mg, about 3mg, about 6mg, about 12mg, about 20mg, about 40mg, about 80mg, about 120mg, about 160mg, about 200mg, about 240mg, and about 300mg ovalbumin.

103. The method of any one of claims 97-102, wherein the dose administered during the upregulating dose phase is only incremented when the patient tolerates the prior dose.

104. The method of any one of claims 97-103, wherein the maximum dose administered to the patient during the upregulating dosage phase is about 300mg egg white protein.

105. The method of any one of claims 97-104, wherein the maintenance dose administered to the patient during the maintenance phase is about 300mg egg white protein or more.

106. The method of any one of claims 97-105, wherein the maintenance dose administered to the patient during the maintenance phase is about 300mg egg white protein.

107. The method of any one of claims 97-106, wherein the maintenance dose is administered to the patient only if the patient tolerates the maximum dose administered to the patient during the upregulating dose phase.

108. The method of any one of claims 97-107, wherein the patient tolerates a dose of about 600mg of raw egg white protein at the end of the maintenance phase.

109. The method of any one of claims 97-108, wherein the patient tolerates a dose of about 1000mg of raw egg white protein at the end of the maintenance phase.

110. The method of any one of claims 97-109, wherein the patient tolerates a dose of about 2000mg of raw egg white protein at the end of the maintenance phase.

111. The method of any one of claims 97-110, wherein the patient tolerates a cumulative dose of about 2000mg mature egg white protein at the end of the maintenance phase.

112. The method of any one of claims 97-111, wherein the patient tolerates a cumulative dose of about 2000mg baked ovalbumin protein at the end of the maintenance phase.

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

114. The method of any one of claims 97-113, wherein the patient is unable to tolerate a cumulative dose of about 2000mg mature egg white protein prior to initiation of treatment.

115. The method of any one of claims 97-114, wherein the patient is unable to tolerate a cumulative dose of about 2000mg baked ovalbumin protein prior to initiation of treatment.

116. The method of any one of claims 97-113, wherein the patient tolerates a cumulative dose of about 2000mg mature egg white protein prior to initiation of treatment.

117. The method of any one of claims 97-113 and 116, wherein the patient tolerates a cumulative dose of about 2000mg baked ovalbumin protein prior to initiation of treatment.

118. The method of any one of claims 97-117, wherein the oral immunotherapy schedule comprises an initial escalation phase prior to the escalation dosage phase, the initial escalation phase comprising orally administering to the patient a series of escalating doses of about 0.2mg to about 2mg of egg white protein over a day, wherein the patient is administered a single administration of any given dose, and wherein the doses are separated by at least 15 minutes.

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

120. The method of any one of claims 97-119, wherein the patient is about 4 years or older prior to initiation of treatment.

121. The method of any one of claims 97-120, wherein the patient is about 4 to about 26 years old before treatment begins.

122. A method of adjusting a dose of a pharmaceutical composition comprising an egg white protein during oral immunotherapy for egg allergy in a subject, the oral immunotherapy comprising (i) an up-dosing phase comprising orally administering a series of ascending doses of the egg white protein to the patient, and (ii) a maintenance phase comprising orally administering a plurality of maintenance doses comprising the egg white protein to the patient; the method comprises the following steps:

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 the second dose is reduced, skipped, or at least part of the dose is delayed if the patient experiences an adverse event related to administration of the first dose.

123. The method of claim 122, wherein if the patient experiences an adverse event related to administration of the first dose, the second dose is divided into a first portion and a second portion, wherein the first portion is administered according to a predetermined dosing schedule, and wherein the second portion is delayed relative to the predetermined dosing schedule.

124. The method of claim 123, wherein the second portion is delayed from about 8 hours to about 12 hours after administration of the first portion.

125. The method of claim 122, wherein the second dose is skipped if the patient experiences an adverse event related to administration of the first dose.

126. The method of claim 122, wherein the second dose is reduced relative to the first dose if the patient experiences an adverse event related to administration of the first dose.

127. The method of claim 126, wherein prior to incrementing a subsequent dose of the pharmaceutical composition, the subsequent dose is reduced relative to the first dose by about one week or more.

128. The method of claim 126 or 127, wherein prior to attempting to increment a subsequent dose of the pharmaceutical composition, the subsequent dose is reduced relative to the first dose by about one week to about two weeks.

129. The method of any one of claims 126 to 128, wherein prior to incrementing a subsequent dose of the pharmaceutical composition, the subsequent dose is reduced relative to the first dose by about one week to about two weeks.

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

131. The method of any one of claims 122-130, wherein the adverse event associated with administration of the first dose is a moderate allergenic adverse event or a severe allergenic adverse event.

132. The method of any one of claims 122-131, wherein the first dose and the second dose are administered to the patient during the up-dosing phase of the oral immunotherapy.

133. The method of any one of claims 122-131, wherein the first dose and the second dose are administered to the patient during the maintenance phase of the oral immunotherapy.

134. A method of adjusting a dose of a pharmaceutical composition comprising an egg white protein during oral immunotherapy for egg allergy in a subject, the oral immunotherapy comprising (i) an up-dosing phase comprising orally administering a series of ascending doses of the egg white protein to the patient, and (ii) a maintenance phase comprising orally administering a plurality of maintenance doses comprising the egg white protein to the patient; the method comprises the following steps:

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 the second dose is reduced or skipped if the patient experiences a complication associated with increased sensitivity to an allergen not associated with administration of the first dose.

135. The method of claim 134, wherein the complication associated with increased sensitivity to an allergen is atopic seizure, inflammation, illness, or menstruation.

136. The method of claim 134 or 135, wherein the second dose is skipped if the patient experiences a complication associated with increased sensitivity to an allergen not associated with administration of the first dose.

137. The method of claim 134 or 135, wherein the second dose is reduced relative to the first dose if the patient experiences a complication associated with increased sensitivity to an allergen not associated with administration of the first dose.

138. The method of claim 137, wherein prior to incrementing a subsequent dose of the pharmaceutical composition, the subsequent dose is reduced relative to the first dose by about one week or more.

139. The method of claim 137 or 138, wherein prior to attempting to increment a subsequent dose of the pharmaceutical composition, the subsequent dose is reduced relative to the first dose by about one week to about two weeks.

140. The method of any one of claims 137 to 139, wherein prior to incrementing a subsequent dose of the pharmaceutical composition, the subsequent dose is reduced relative to the first dose by about one week to about two weeks.

141. The method of any one of claims 134-140, wherein the first dose and the second dose are administered to the patient during the up-dosing phase of the oral immunotherapy.

142. The method of any one of claims 134-141, wherein the first dose and the second dose are administered to the patient during the maintenance phase of the oral immunotherapy.

143. The method of any one of claims 134 to 142, wherein a complication associated with increased sensitivity to an allergen is unintended exposure of the patient to food to which the patient is allergic.

144. The method of any one of claims 97-143, wherein the egg white protein in the pharmaceutical composition is a raw egg white protein.

145. The method of any one of claims 97-144, wherein the pharmaceutical composition is mixed with a food vehicle prior to administration.

146. A pharmaceutical composition for use in a method according to any one of claims 97 to 145.

147. The pharmaceutical composition of claim 146, wherein the pharmaceutical composition comprises an egg white protein formulation prepared according to the method of any one of claims 1 to 96.

148. A pharmaceutical composition for use in the preparation of a medicament for use in a method of treating egg allergy in a patient according to the method of any one of claims 97 to 145.

149. The pharmaceutical composition of claim 148, wherein the pharmaceutical composition comprises an egg white protein formulation prepared according to the method of any one of claims 1-96.

Technical Field

The present invention relates to formulations for egg oral immunotherapy and methods of preparing such formulations, and to oral immunotherapy treatments for egg allergy in patients.

Background

IgE-mediated egg allergy is one of the most common food allergies in childhood and can be associated with severe immediate hypersensitivity reactions, such as life-threatening allergies. The incidence of egg allergy in western countries is estimated to be about 0.5% to 2.5%. Although most children often develop egg allergy over time, longitudinal studies indicate that this may not occur until the second decade of life, where egg allergy persists in about 20% of individuals aged 18 years.

Two major egg allergy phenotypes are clinically identified: (1) allergy to both egg white and baked whole egg, and (2) allergy to egg white alone. About 20% to 30% of egg allergic individuals are allergic to both egg white and baked whole eggs, and 70% to 80% of egg allergic individuals are allergic to only egg white.

The current standard of care for managing egg allergy is diet avoidance of eggs and training patients/families to identify and manage allergy symptoms and general use of emergency medications, such as epinephrine auto-injectors (EAIs). However, it is particularly difficult for egg allergy sufferers to avoid eggs, as eggs are ubiquitous as an ingredient in many food products and are also very important as a childhood nutrient. The burden of avoidance and the continued fear of accidental exposure can adversely affect the health-related quality of life of individuals with egg allergy and their families. Complicating medical need, the daily carriage and emergency use of EAI for the treatment of allergies is considered inadequate, which may lead to adverse consequences including hospitalization and death.

It is generally believed that ingestion of milligram quantities of egg protein alone may trigger allergic reactions based on studies with an estimated population threshold. Although the threshold level at which allergic responses are triggered varies widely throughout the egg allergy population, the ED10 (the priming dose expected to elicit a response in 10% of individuals with a particular food allergy) is estimated to be in the range of 3.7mg to 5.8mg egg protein. Therefore, accidental allergic reactions to eggs are common.

Currently available therapies are designed to treat only the symptoms of an allergic reaction when an allergic reaction occurs. Thus, therapies with the potential to reduce the risk of severe allergic reactions in the event of accidental exposure, without cure, represent an urgent unmet medical need. However, developing carefully calibrated dosage forms for delivery of egg allergens by oral immunotherapy remains a challenge, particularly for lower dosage forms. For lower doses, control of the allergenic material in oral immunotherapeutic doses is particularly important to limit adverse events associated with the therapy. Therefore, batch manufacturing processes, especially for low dose manufacturing, should have sufficient uniformity throughout the blended product to ensure adequate dose uniformity in the packaged product.

In recent years, Oral Immunotherapy (OIT) for egg allergy has been studied, which involves the oral administration of allergenic proteins to patients in increasing doses to obtain a desensitized state. However, further improvements in the safety and/or efficacy of treatment are desired.

Disclosure of Invention

Described herein are egg white protein formulations, dosage forms comprising egg white protein formulations, and methods of making such egg white protein formulations and dosage forms.

In some embodiments, a method of preparing an egg white protein formulation comprises: (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 at a higher shear force than the shear force used for mixing in step (c); and (f) mixing the third mixture with a lubricant to form an egg white protein formulation. In some embodiments, the egg white protein formulation has about 0.05% to about 2.5% egg white protein by weight. In some embodiments, the egg white protein formulation has about 0.1% to about 0.7% egg white protein by weight. In some embodiments, step (b) comprises passing at least a portion of the second amount of the first diluent with the first mixture through a mesh screen. In some embodiments, step (b) comprises passing the second amount of the first diluent with the first mixture through a mesh screen. In some embodiments, step (c) comprises a plurality of sub-steps, wherein each sub-step comprises (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 with the first mixture. In some embodiments, step (c) comprises three or more substeps. In some embodiments, step (c) comprises continuously mixing the first mixture and the second amount of the first diluent while the second amount of the first diluent is added to the first mixture. In some embodiments, the first mixture is mixed with a second amount of the first diluent in a drum blender. In some embodiments, the second mixture is mixed with a second diluent in a drum blender. In some embodiments, the third mixture is mixed using a cone mill. In some embodiments, the third mixture is mixed with the lubricant in a drum 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 and the lubricant to form the egg white protein formulation. In some embodiments, the egg white protein preparation is substantially free of colloidal silica. In some embodiments, the egg white protein formulation has about 40% to about 70% by weight of the first diluent. In some embodiments, the egg white protein formulation has about 30% to about 50% by weight of the second diluent. In some embodiments, the egg white protein formulation has from about 0.1% to about 2% by weight of a lubricant.

In some embodiments, a method of preparing an egg white protein formulation comprises (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 the shear force used for mixing in step (b); and (e) mixing the third mixture with a lubricant to form the egg white protein formulation. In some embodiments, the dried egg white protein powder is mixed with a first amount of a first diluent in step (a) at a higher shear force than the shear force used for mixing in step (b). In some embodiments, the egg white protein formulation has about 1% to about 70% egg white protein by weight. In some embodiments, the dried egg white protein powder is mixed with a first amount of a first diluent in step (a) using a cone mill. In some embodiments, step (b) comprises two mixing sub-steps, wherein one mixing sub-step is at a higher shear force than the other mixing sub-step. In some embodiments, step (c) 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. In some embodiments, step (c) further comprises co-mixing the second mixture with additional amounts of the first diluent and the second diluent to form a third mixture. In some embodiments, the third mixture is mixed in step (d) using a cone mill. In some embodiments, the second amount of the first diluent and the first mixture are mixed in a drum blender. In some embodiments, the second mixture is mixed with a second diluent in a drum 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 (e) comprises (i) mixing a portion of the third mixture with a lubricant; (ii) (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. In some embodiments, the third mixture is mixed with the lubricant in a drum blender. In some embodiments, the egg white protein formulation has about 9% to about 85% by weight of the first diluent. In some embodiments, the egg white protein formulation has from about 10% to about 50% by weight of the second diluent. In some embodiments, the egg white protein formulation has from about 10% to about 20% by weight of the second diluent. In some embodiments, the egg white protein formulation has from about 0.1% to about 2% by weight of a lubricant. In some embodiments, the egg white protein formulation comprises a glidant. In some embodiments, during or prior to step (a), the glidant is mixed with the egg white protein powder and the first amount of the first diluent. In some embodiments, the glidant is colloidal silicon dioxide. In some embodiments, the egg white protein formulation has about 2% to about 70% egg white protein by weight. In some embodiments, the egg white protein formulation is substantially free of colloidal silica and may comprise from about 1% to about 5% by weight egg white protein.

In another embodiment, a method of making an egg white protein formulation comprises: (a) mixing a dry egg white protein powder, a first diluent, and a glidant to form a first mixture; (b) mixing a second diluent with the first mixture at a higher shear force than the shear force used for mixing in step (a) to form a second mixture; (c) the second mixture is mixed with a lubricant to form an egg white protein formulation. In some embodiments, the egg white protein formulation has about 50% to about 80% egg white protein by weight. In some embodiments, the dried egg white protein powder, the first diluent, and the glidant are mixed in a drum blender. In some embodiments, the second diluent and the first mixture are mixed in step (b) using a cone mill. In some embodiments, the second mixture and the lubricant are mixed in a drum 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 the lubricant are co-mixed with the second mixture. In some embodiments, the glidant comprises colloidal silicon dioxide. In some embodiments, 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 the mixture of (ii) with another portion of the second mixture to form an egg white protein preparation.

In some embodiments of the above methods, the egg white protein preparation is prepared in a batch size of about 5kg or greater. In some embodiments, the egg white protein formulation is prepared in a batch size of about 5kg to about 50 kg.

In some embodiments of the above method, the method further comprises determining an egg white protein blend homogeneity of the egg white protein formulation. In some embodiments, the egg white protein formulation has an egg white protein blend uniformity Relative Standard Deviation (RSD) of about 15% or less.

In some embodiments of the above method, the method further comprises packaging the egg white protein preparation in a plurality of dosage containers. In some embodiments, the dosage container is a capsule or a sachet. In some embodiments, the method further comprises determining an egg white protein content uniformity of the plurality of dosage containers. In some embodiments, the egg white protein content uniformity relative standard deviation (RDS) of the plurality of dosage containers is about 15% or less.

In some embodiments of the above method, the first diluent is a pregelatinized starch.

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

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

In some embodiments of the above methods, the dried egg white protein powder comprises about 50% to about 90% by weight egg white protein.

In some embodiments of the above methods, forming the dried egg white protein powder comprises spray drying the liquid egg white.

In some embodiments of the above methods, the egg white protein powder has been depleted of glucose.

In some embodiments of the above methods, the dried egg white powder has been pasteurized.

In some embodiments of the above methods, the dried egg white protein powder is derived from an egg.

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

In some embodiments of the above method, the method further comprises characterizing the ovomucoid, ovalbumin, ovotransferrin, or lysozyme in the egg white protein formulation.

In some embodiments, characterizing the 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.

In some embodiments, characterizing the ovomucoid, ovalbumin, ovotransferrin, or lysozyme comprises quantifying the amount of the 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 compared to the total protein in the ovalbumin powder or ovalbumin preparation. In some embodiments, quantifying the amount of ovomucoid, ovalbumin, ovotransferrin, or lysozyme comprises measuring the amount of ovomucoid, ovalbumin, ovotransferrin, or lysozyme compared to the total amount of ovomucoid, ovalbumin, ovotransferrin, or lysozyme in the ovalbumin powder or ovalbumin formulation.

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

Also described herein are egg white protein formulations prepared according to any of the above methods.

In some embodiments, an egg white protein formulation comprises 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 silica. In some embodiments, the egg white protein formulation comprises about 0.1% to about 3.5% egg white protein by weight. 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.

Also described herein are methods of treating egg allergy in a patient, and methods of adjusting the dose of a pharmaceutical composition comprising egg white protein during oral immunotherapy for egg allergy.

In one embodiment, a method of treating egg allergy in a patient comprises orally administering to the patient a plurality of doses of a pharmaceutical composition comprising an egg white protein according to an oral immunotherapy schedule comprising: (a) an up-dosing phase comprising orally administering to the patient a series of escalating doses of about 1mg to about 300mg egg white protein, wherein a given dose is administered to the patient for at least two weeks prior to dose escalation, and wherein the length of the up-dosing phase is about 20 weeks to about 44 weeks; and (b) a maintenance phase comprising orally administering to the patient a plurality of maintenance doses comprising egg white protein, wherein the maintenance phase is about 12 weeks or more in length.

In some embodiments, the patient has about 7kU at the start of treatment_A(ii) egg white-specific serum IgE (ew-IgE) levels of/L or higher. In some embodiments, the patient has about 5kU at the start of treatment_A(ii) egg white-specific serum IgE (ew-IgE) levels of/L or higher.

In some embodiments, the series of escalating doses administered to the patient during the up-dosing phase includes at least a 1mg dose of egg white protein and a 300mg dose of egg white protein. In some embodiments, the series of escalating doses administered to the patient during the up-dosing phase includes at least 10 different doses of egg white protein. In some embodiments, the series of escalating doses administered to the patient during the up-dosing phase includes doses of egg white protein of about 1mg, about 3mg, about 6mg, about 12mg, about 20mg, about 40mg, about 80mg, about 120mg, about 160mg, about 200mg, about 240mg, and about 300 mg.

In some embodiments, the dose administered during the up-dosing phase is only incremented when the patient tolerates the previous dose.

In some embodiments, the maximum dose administered to the patient during the up-dosing phase is about 300mg of egg white protein.

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

In some embodiments, the maintenance dose is administered to the patient only if the patient tolerates the maximum dose administered to the patient during the up-dosing phase.

In some embodiments, the patient tolerates a dose of about 600mg of raw egg white protein at the end of the maintenance phase. In some embodiments, the patient tolerates a dose of about 1000mg of raw egg white protein at the end of the maintenance phase. In some embodiments, the patient tolerates a dose of about 2000mg of raw egg white protein at the end of the maintenance phase. In some embodiments, the patient tolerates a cumulative dose of about 2000mg of mature egg white protein at the end of the maintenance phase. In some embodiments, the patient tolerates a cumulative dose of about 2000mg of baked egg white protein at the end of the maintenance phase.

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

In some embodiments, the oral immunotherapy schedule comprises an initial escalation phase prior to the escalation dosage phase, the initial escalation phase comprising orally administering to the patient a series of escalated doses of about 0.2mg to about 2mg of egg white protein within a day, wherein a single administration of any given dose is administered to the patient, and wherein the doses are separated by at least 15 minutes.

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

In some embodiments, the patient is about 4 years or older before treatment begins. In some embodiments, the patient is about 4 years to about 26 years old before treatment begins.

Also described herein are methods of adjusting a dose of a pharmaceutical composition comprising egg white protein during oral immunotherapy for egg allergy in a subject, the oral immunotherapy comprising (i) an up-dosing phase comprising orally administering a series of ascending doses of egg white protein to a patient, and (ii) a maintenance phase comprising orally administering a plurality of maintenance doses comprising egg white protein to a patient; the method comprises the following steps: orally administering a first dose of the pharmaceutical composition to a patient; and orally administering a second dose of the pharmaceutical composition to the patient, wherein the second dose is reduced, skipped, or at least a portion of the dose is delayed if the patient experiences an adverse event associated with administration of the first dose. In some embodiments, if the patient experiences an adverse event associated with administration of the first dose, the second dose is divided into a first portion and a second portion, wherein the first portion is administered according to a predetermined dosing schedule, and wherein the second portion is delayed relative to the predetermined dosing schedule. In some embodiments, the second part is delayed from about 8 hours to about 12 hours after administration of the first part. In some embodiments, the second dose is skipped if the patient experiences an adverse event related to administration of the first dose. In some embodiments, the second dose is reduced relative to the first dose if the patient experiences an adverse event associated with administration of the first dose. In some embodiments, prior to incrementing the subsequent dose of the pharmaceutical composition, the subsequent dose is reduced relative to the first dose by about one week or more. In some embodiments, prior to attempting to increment a subsequent dose of the pharmaceutical composition, the subsequent dose is reduced relative to the first dose by about one week to about two weeks. In some embodiments, prior to incrementing the subsequent dose of the pharmaceutical composition, the subsequent dose is reduced relative to the first dose by about one week to about two weeks. 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 the up-dosing phase of oral immunotherapy. In some embodiments, the first dose and the second dose are administered to the patient during a maintenance phase of oral immunotherapy.

Also described herein are methods of adjusting a dose of a pharmaceutical composition comprising egg white protein during oral immunotherapy for egg allergy in a subject, the oral immunotherapy comprising (i) an up-dosing phase comprising orally administering a series of ascending doses of egg white protein to a patient, and (ii) a maintenance phase comprising orally administering a plurality of maintenance doses comprising egg white protein to a patient; the method comprises the following steps: orally administering a first dose of the pharmaceutical composition to a patient; and orally administering a second dose of the pharmaceutical composition to the patient, wherein the second dose is reduced or skipped if the patient experiences complications associated with increased sensitivity to an allergen not associated with administration of the first dose. In some embodiments, the complication associated with increased sensitivity to an allergen is atopic seizure, inflammation, illness, or menstruation. In some embodiments, the second dose is skipped if the patient experiences a complication associated with increased sensitivity to an allergen not associated with administration of the first dose. In some embodiments, if the patient experiences a complication associated with increased sensitivity to an allergen not associated with administration of the first dose, the second dose is reduced relative to the first dose. In some embodiments, prior to incrementing the subsequent dose of the pharmaceutical composition, the subsequent dose is reduced relative to the first dose by about one week or more. In some embodiments, prior to attempting to increment a subsequent dose of the pharmaceutical composition, the subsequent dose is reduced relative to the first dose by about one week to about two weeks. In some embodiments, prior to incrementing the subsequent dose of the pharmaceutical composition, the subsequent dose is reduced relative to the first dose by about one week to about two weeks. In some embodiments, the first dose and the second dose are administered to the patient during the up-dosing phase of oral immunotherapy. In some embodiments, the first dose and the second dose are administered to the patient during a maintenance phase of oral immunotherapy. In some embodiments, a complication associated with increased sensitivity to an allergen is unintended exposure of the patient to their allergic food.

In some embodiments of the above methods, the egg white protein in the pharmaceutical composition is a native egg white protein.

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

Also provided herein is a pharmaceutical composition for use in a method of treating egg allergy as described above, wherein the pharmaceutical composition comprises an egg white protein formulation prepared according to the aforementioned method of preparing an egg white protein formulation.

Also provided herein is a pharmaceutical composition for use in the manufacture of a medicament for use in a method of treating egg allergy in a patient according to the above method of treating egg allergy, wherein the pharmaceutical composition comprises an egg white protein formulation prepared according to the aforementioned method of preparing an egg white protein formulation.

Drawings

Figure 1 illustrates an exemplary method of preparing an egg white protein formulation, particularly for lower dosage levels.

Figure 2 shows an exemplary method of preparing an egg white protein formulation, particularly for moderate dose levels.

Figure 3 shows an exemplary method of preparing an egg white protein formulation, particularly for higher dose levels.

Fig. 4 shows an exemplary decision tree for adjusting the dose according to the severity of adverse events associated with administration of ovalbumin during oral immunotherapy.

Figure 5 shows a size exclusion chromatography HPLC chromatogram of a dried egg white protein powder with identified egg white proteins, ovomucoid, ovotransferrin and lysozyme.

Figure 6 shows a reverse phase HPLC chromatogram of dried egg white protein powder with identified egg white proteins, ovomucoid, ovotransferrin and lysozyme.

FIG. 7 shows SDS-PAGE gels of dried egg white protein powder (lanes 3-5; about 2. mu.g protein/well) and protein standards (ovomucoid, ovalbumin, lysozyme and ovotransferrin; lanes 7-10).

FIG. 8 shows immunoblots of dried egg white protein powder (lanes 3-5) and protein standards (ovomucoid, ovalbumin, lysozyme and ovotransferrin; lanes 7-10) stained to identify ovalbumin, ovomucoid, ovotransferrin and lysozyme.

Figure 9 shows a schematic of a clinical study of a pharmaceutical composition comprising an egg white protein.

Figure 10 shows a schematic of a clinical study of a pharmaceutical composition comprising an egg white protein.

FIG. 11 shows a reverse phase HPLC chromatogram of a dry egg white protein powder using a modified RP-HPLC method in which relative retention time peaks of ovomucoid, lysozyme, ovotransferrin and egg white protein were identified.

Fig. 12A shows an exemplary method of preparing an egg white protein formulation, particularly for lower dose levels.

Figure 12B illustrates another exemplary method of preparing an egg white protein formulation, particularly for lower dose levels.

Fig. 12C shows another exemplary method of preparing an egg white protein formulation, particularly for lower dose levels.

Fig. 13A shows an exemplary method of preparing an egg white protein formulation, particularly for intermediate dose levels.

Fig. 13B shows another exemplary method of preparing an egg white protein formulation, particularly for intermediate dose levels.

Fig. 13C shows another exemplary method of preparing an egg white protein formulation, particularly for intermediate dose levels.

Fig. 14A shows an exemplary method of preparing an egg white protein formulation, particularly for higher dose levels.

Figure 14B illustrates another exemplary method of preparing an egg white protein formulation, particularly for higher dose levels.

Detailed Description

Described herein are egg white protein formulations and methods of making such egg white protein formulations. Egg white protein formulations comprise dried egg white protein powder, together with one or more diluents, lubricants and (in some formulations) glidants, and are useful for treating egg allergy by oral immunotherapy. Since highly allergic patients may be susceptible to minor variations in the amount of allergen contained in the formulation, it is desirable to carefully calibrate the amount and quality of egg allergen contained in the formulation, for example by using the quality control and/or manufacturing processes described herein. The preparation methods described herein allow for the production of egg white protein formulations with carefully controlled amounts of egg allergens.

A significant challenge in preparing egg white protein formulations is the expansion of batch sizes. Uniformity in batches on smaller research platforms is more readily available than large scale preparations for commercial production. Obtaining a sufficiently homogeneous formulation blend is particularly challenging and important for low dose formulations, where patients consuming such doses may be particularly sensitive to egg white allergens. The manufacturing process described herein provides for an expansion of the manufacturing of egg white protein preparations, including batches of greater than 5 kg.

In some methods of making an egg white protein preparation, the method comprises (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 at a higher shear force than the shear force used for mixing in step (c); and (f) mixing the third mixture with a lubricant to form an egg white protein formulation. In step (e), the third mixture is mixed to dissociate the particle agglomerates. Step (c) may be performed in a plurality of sub-steps to serially dilute the mixture with two or more different portions of the second amount of the first diluent. This method of preparing a formulation is particularly useful for preparing formulations for use in lower dose dosage containers containing the formulation, such as doses of about 0.1mg to about 12 mg. The formulation may have, for example, about 0.05% to about 2.5% by weight egg white protein. Glidants (e.g., colloidal silicon dioxide) need not be included in lower dose formulations.

In some methods of making an egg white protein formulation, the method comprises (a) mixing a dry egg white protein powder, a first diluent, and a glidant to form a first mixture; (b) mixing a second diluent with the first mixture at a higher shear force than the shear force used for mixing in step (a) to form a second mixture; (c) the second mixture is mixed with a lubricant to form an egg white protein formulation. Mixing in step (b) is carried out such that the mixing dissociates the particle agglomerates. This method of preparing a formulation is particularly useful for preparing formulations containing higher dose dosage containers of the formulation, such as doses of about 200mg and greater, such as up to 600mg, or up to 300 mg. The formulation may have, for example, about 50% to about 80% by weight egg white protein. The egg white protein formulations described herein comprise, consist essentially of, or consist of: a dry egg white protein powder, one or more diluents (e.g., two diluents), and a lubricant. In some embodiments, the egg white protein formulation is substantially free of glidants or substantially free of colloidal silicon dioxide. Optionally, the egg white protein preparation comprises, consists essentially of, or consists of: a dry egg white protein powder, one or more diluents (e.g., two diluents), a lubricant, and a glidant (such as colloidal silicon dioxide).

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

The dry egg white protein powder used to prepare the formulation, the prepared egg white protein formulation and the prepared dosage container containing the egg white protein formulation should be carefully controlled to ensure accurate administration of the egg white protein dosage to patients to be treated for egg allergy by oral immunotherapy. As further described herein, one or more methods can be used to assess the quality of the dried egg white protein powder, egg white protein formulation, or dosage container. Such methods include measuring the protein content of the powder or formulation, characterizing one or more allergenic egg white proteins in the powder or formulation (e.g., ovalbumin, ovomucoid, ovotransferrin, and/or lysozyme), determining blend uniformity of the formulation, measuring deliverable amounts of the formulation from bulk dose containers, or measuring content uniformity in bulk dose containers.

Also described herein are methods of treating egg allergy in a patient by orally administering to the patient a plurality of doses of a pharmaceutical composition comprising egg white protein according to an oral immunotherapy schedule comprising (a) an up-dosing phase comprising orally administering to the patient a series of ascending doses of about 1mg to about 300mg egg white protein, wherein a given dose is administered to the patient for at least two weeks prior to dose escalation, and wherein the up-dosing phase is about 20 weeks to about 44 weeks in length; and (b) a maintenance phase comprising orally administering to the patient a plurality of maintenance doses comprising egg white protein, wherein the maintenance phase is about 12 weeks or more in length.

The dose of the pharmaceutical composition administered to the patient may be adjusted (e.g., by reducing the dose, skipping the dose, delaying (all or part) of the dose administration, or delaying the dose increase) in response to an adverse event associated with administration of the pharmaceutical composition or some other complication associated with increased sensitivity to an allergen not associated with administration of the pharmaceutical composition to the patient, such as atopic disease onset, inflammation, disease, menstruation, or unintended exposure of the patient to food to which the patient is allergic.

The Oral Immunotherapy (OIT) treatment schedule described herein is intended to safely desensitize patients who are allergic to egg proteins, enabling the patients to ingest egg proteins through accidental exposure without moderate or severe allergenic adverse events. Depending on the individual patient's treatment outcome, the patient may remain protected from eating cooked eggs after treatment.

Definition of

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

Reference herein to "about" a value or parameter includes (and describes) variations that relate to that value or parameter itself. For example, a description referring to "about X" includes a description of "X".

An "adverse event" is any adverse and unexpected sign (including abnormal laboratory findings), symptom or disease, or exacerbation thereof, temporally associated with oral immunotherapy, whether or not associated with oral immunotherapy.

Compositions "consisting essentially of" the listed materials include those materials that constitute only at least 99.95% by weight of the composition. Additives, impurities, or other components may comprise less than 0.05% by weight of the composition.

The term "cooked" refers to heat treating a composition without combusting the composition, which results in at least partial modification or denaturation of one or more proteins in the composition. The term "baking" refers to heat treating the composition at a temperature of at least 176 ℃ for at least 20 minutes without burning the composition.

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

Unless otherwise indicated, the terms "effective," "efficacy," or "effectiveness" are used herein to refer to the ability of a therapy to induce immune modulation (such as desensitization) or to maintain a desired immune state (such as a desensitized state).

An "ascending dose" is a first dose administered to a patient that is higher than an immediately previous dose administered to the patient during the course of oral immunotherapy.

As used herein, "maintenance phase" refers to the phase of egg protein oral immunotherapy that includes administration of egg white protein to a patient (i.e., maintenance dose) and occurs upon completion of the up-dosing phase.

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

As used herein, "moderate allergic adverse event" refers to an allergic adverse event associated with discomfort of a sufficient extent to interfere with daily activities that is observed or experienced and may be suggestive of medical intervention and/or otherwise observed OIT treatment-related allergic adverse events.

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

The term "subject" or "patient" is used synonymously herein to describe a human of any age.

A composition is "substantially free" of material if it contains less than 0.005 weight percent of the material or is free of the material.

A dose is "tolerated" by a patient when it is administered to and consumed completely by the patient without any moderate or severe allergic adverse events in response to the dose. Patients are considered to tolerate this dose even if mild allergic adverse events are observed or experienced. By "highest tolerated dose" is meant the maximum dose that a patient administered to the patient during an oral food challenge is tolerant without any moderate or severe allergic adverse events. The "cumulative tolerated dose" is the sum of the doses administered to the patient up to and including the highest tolerated dose without any moderate or severe adverse allergic events during the oral food challenge.

The terms "treat," "treating," and "treatment" are used synonymously herein and refer to any action that provides a beneficial effect to a subject afflicted with a disease state or disorder, including ameliorating the disorder by reducing, inhibiting, suppressing, or eliminating at least one symptom; delay of disease progression; delay in disease recurrence; inhibiting the disease; or partially or completely reduce the response or reaction to an allergen.

By "up-dosing phase" is meant a phase of oral immunotherapy characterized by a series of increasing food allergen doses beginning with a food allergen dose that is lower than the highest dose administered to the patient during oral immunotherapy and ending when the highest dose administered to the patient during oral immunotherapy is achieved.

The terms "weight percent," "weight percent," and "weight%" are used synonymously herein and refer to the percentage of the listed components in the composition relative to the total weight of the composition.

It should be understood that aspects and variations of the invention described herein include aspects and variations that "consist of and/or" consist essentially of.

Where a range of values is provided, it is understood that each intervening value, to the extent that there is no such stated or intervening value in the stated range, is encompassed within the scope of the disclosure. Where the stated range includes an upper limit or a lower limit, ranges excluding any of those included limits are also included in the disclosure.

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

The features and preferences described above in connection with the "embodiment" are various preferences and are not limited to only this particular embodiment; they can be freely combined with features from other embodiments, where technically feasible, and can form preferred combinations of features. This description is provided to enable one of ordinary skill 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 herein 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.

The disclosures of all publications, patents and patent applications mentioned herein are hereby incorporated by reference in their entirety. To the extent any reference incorporated by reference conflicts with the present disclosure, the present disclosure shall control.

Egg white protein formulations

The egg white protein formulations described herein comprise a Dried Egg White (DEW) protein powder and may be formulated with one or more excipients. Typically, the formulation comprises one or more different diluents (preferably two different diluents) and a lubricant. Some egg white protein formulations also contain a glidant, but a glidant may be omitted in some formulations. For example, glidants are typically omitted in lower dose formulations (e.g., formulations having less than about 2.5 wt% ovalbumin). Thus, in some formulations, the egg white protein formulation is substantially free of glidants or substantially free of colloidal silicon dioxide. When packaged in a dosage container, the excipients and the ratio of excipients may be selected according to the dosage of egg white protein.

The dried egg white protein powder is typically derived from poultry, preferably from chicken (Gallus poultry) egg white. Dried egg white protein powder is often used as a food ingredient, although it may be formulated for use as an oral immunotherapy formulation for the treatment of egg allergy. Oral immunotherapy treatment using unformulated dry egg white protein powder is undesirable because safe treatment requires careful dosing, especially at lower doses, which is difficult to achieve consistently without formulation. The dry egg white protein powder typically has a protein content of about 80% to about 85%, but the amount of dry egg white protein powder added to the formulation batch may be adjusted based on the actual protein content of the dry egg white protein powder, while adjusting the amount of one or more excipients (such as one of these diluents, such as pregelatinized starch) to achieve the desired dosage level. For example, the protein content of the dried egg white protein powder can be between about 50% and about 90% by weight (such as about 70% to about 90%, about 75% to about 90%, or about 70% to about 85%). The remaining weight of the dried egg white protein powder may include small amounts of fat, carbohydrates, vitamins, minerals, or other components naturally present in egg white. The dried egg white protein powder may also contain residual water.

Dry egg white protein powder is typically derived from liquid egg white by spray drying the dry egg white. Preferably, the dried egg white protein powder is pasteurized or sterilized (e.g., by irradiation), but care should be taken so that the allergenic protein is not denatured during the manufacturing process. While spray drying and pasteurization are not expected to denature proteins, quality control procedures (such as determining 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 egg white protein preparation produced. Degradation of lysozyme or ovotransferrin can also be detected using polyacrylamide gel electrophoresis (PAGE) (e.g., SDS-PAGE) or immunoblotting, and when degraded, the lysozyme or ovotransferrin disappears or fades from the gel.

Dry egg white protein powder can be prepared by separating liquid egg white and egg yolk from broken eggs. If desired, the pH of the egg white can be adjusted using a suitable acid or base as is known in the art. Glucose can be removed from liquid egg white, which can reduce the browning of dry egg white protein powder that can occur through maillard reactions. Glucose can be removed, for example, by adding a solution of glucose oxidase, catalase, and hydrogen peroxide to liquid egg white or by fermentation (e.g., by adding yeast to the egg white).

To dry the liquid egg white, the liquid egg white may be passed through a spray dryer. Liquid egg white is atomized into the chamber by a spray dryer nozzle and dried using a stream of hot air. The amount of heat and exposure time are set to dry the liquid egg white to a powder, but without denaturing the allergenic proteins within the egg white (e.g., lysozyme, ovalbumin, ovomucoid, and ovotransferrin).

The dried egg white protein powder may be pasteurized after the drying process. Pasteurization kills harmful bacteria such as Salmonella (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 may be held at about 50 ℃ to about 60 ℃ (and preferably at about 54 ℃ to about 60 ℃) for about 7 days or longer. The pasteurization process may continue at least until the dry egg white powder is negative for live salmonella and/or e.

The dry egg white protein powder may include materials added during the powder processing process, such as glucose oxidase, catalase, hydrogen peroxide, acids or bases for adjusting pH, or vehicles for any of the components (e.g., salts, buffers, stabilizers, etc.). However, these additives are relatively few, and it is generally preferred that the dried egg white protein powder be substantially free of additives prior to preparation of the egg white protein formulation, wherein the formulation excipients are mixed with the dried egg white protein powder.

Since the egg white protein formulations were prepared by combining and blending powders, the particle size of the dried egg white protein powder was identified as a potential factor that could affect the blend uniformity of the mass produced formulations and/or the content uniformity of the batch of dosage containers, especially at the lowest dosage intensity. The powder particle size can affect the blending process, the adherence of the particles to the carrier vehicle or processing equipment surface, or segregation after the blending process. Median particle size (D) of particles in dry egg white protein powder for use in the formulations described herein₅₀) Typically from about 30 μm to about 150 μm (such as from about 30 μm to about 50 μm, from about 50 μm to about 80 μm, from about 80 μm to about 120 μm, or from about 120 μm to about 150 μm). D₁₀Typically about 3 μm or more (and less than the median particle diameter). D₉₀Typically about 250 μm or less (and greater than the median particle diameter), such as about 200 μm or less, or about 175 μm or less. Particle size distribution metric (D)₅₀、D₉₀、D₁₀) Refers to the particle size at the reference percentile (i.e., 50 th, 90 th, or 10 th) in cumulative mass (or cumulative volume, which is equivalent to the mass of a uniformly dense particle) or in cumulative number. The particle size can be measured using standard laser diffraction techniques (e.g., dynamic laser light scattering), in which the particles are suspended in a solvent that does not dissolve the particles, such as methanol, ethanol, or other suitable solvent, or by sieve analysis.

The excipients included in an egg white protein formulation are selected so that the formulation can be packaged (e.g., in a capsule, sachet, or other suitable package) at an accurate and consistent level of egg white protein. The excipients of the formulation include one or more (and preferably two) different diluents and lubricants. In some formulations, a glidant is included. However, in some embodiments, the glidant is omitted from the formulation. For example, in some formulations (such as lower dose formulations having less than about 5 wt.% egg white protein), the egg white protein formulation is free or substantially free of colloidal silica. Exemplary 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 typically included in the formulation. If included in the formulation, the glidant is preferably colloidal silicon dioxide.

The egg white protein formulation is packaged into a dosage container, and the amount of the egg white protein formulation and the concentration of egg white protein in the formulation determine the dosage of egg white protein in the dosage container. The dosage container is not consumed in its entirety, but the contents are removed prior to consumption. Once removed, the egg white protein preparation may be mixed with a food vehicle (such as yogurt, oatmeal, pudding, apple sauce, or other suitable food vehicle) to aid in consumption. Because dosage accuracy is particularly important when administering oral immunotherapy, the egg white protein formulation should have good flow characteristics to ensure that a sufficient portion of the container contents are removed and consumed. The formulations described herein allow the egg white protein formulation in the container to flow sufficiently out of the container prior to consumption.

Pregelatinized starch and microcrystalline cellulose are particularly useful diluents because they are free-flowing powders that are mixed with egg white protein powder. A lower egg white protein dosage formulation (e.g., a dose of about 10mg or less) contains a higher percentage of microcrystalline cellulose than a higher dose, as this ensures good flowability and bulk density of the egg white protein formulation for packaging in and removal from a container, such as packaging in or removal from a capsule. For example, a lower dose (e.g., about 10mg or less) of an egg white protein formulation may comprise about 25% to about 60% (such as about 25% to about 30%, about 30% to about 40%, about 40% to about 50%, or about 50% to about 60%) by weight microcrystalline cellulose, while a higher dose (e.g., greater than about 10mg) of an egg white protein formulation may comprise about 5% to about 25% (such as about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, or about 20% to about 25%) by weight.

Magnesium stearate was found to be a useful lubricant to prevent egg white protein from adhering to equipment surfaces during the manufacturing process. Generally, an egg white protein formulation comprises from about 0.2% to about 2% by weight, such as from about 0.2% to about 0.4%, from about 0.4% to about 0.6%, from about 0.6% to about 1%, from about 1% to about 1.5%, or from about 1.5% to about 2% by weight magnesium stearate. In some embodiments, the egg white protein formulation comprises about 0.5% by weight magnesium stearate.

Glidants such as colloidal silicon dioxide may be included in egg white protein formulations at higher doses (e.g., doses over about 6mg egg white protein or doses over about 12mg egg white protein). It was found that the omission of glidants in larger doses resulted in part of the ovalbumin formulation remaining in the container, which would result in underdosing of the patient. However, the inclusion of a glidant in an egg white formulation allows substantially all of the egg white protein formulation to be delivered from the container upon decanting. The addition of colloidal silica was found to form soft, low density agglomerates. A high shear mixing step (such as a cone mill) is a solution that has been found to improve the content uniformity of formulations containing colloidal silica. Preferably, the high shear mixing step that disrupts these low density agglomerates does not alter the primary particle size of any of the blend components. Thus, a glidant, such as colloidal silicon dioxide, may be included in an egg white protein formulation in an amount of, for example, about 0.2% to about 2% by weight (such as about 0.2% to about 0.4%, about 0.4% to about 0.6%, about 0.6% to about 1%, about 1% to about 1.5%, or about 1.5% to about 2%). Inclusion of a glidant may increase the deliverability of the egg white protein powder from the container in higher doses. In some embodiments, about 95% or more by weight (such as about 96% or more, about 97% or more, about 98% or more, about 99% or more, or about 99.5% or more) of the egg white protein formulation in the container can be delivered from the container. In addition, 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 a plurality of dosage containers, and the average deliverable egg white protein formulation is 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).

For lower dose egg white protein preparations (e.g., about 6mg egg white protein or less), silica was found to interfere with quality control analysis, and in particular to quantify protein concentration. As discussed previously, the amount of egg white protein contained in the dosage container should be accurate to minimize the risk of accidental overdose, especially when low doses are administered to the subject. It has also been found that glidants included in egg white protein formulations at higher dosage amounts to ensure adequate deliverability from the dosage container can be omitted in egg white protein formulations used at lower dosage amounts while maintaining adequate deliverability from the container. In some embodiments, even when the egg white protein formulation is free of colloidal silica, 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 a dosage container comprising 6mg egg white protein or less (such as between about 0.1mg and about 6mg egg white protein) may be delivered from the container. In addition, 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 a plurality of dosage containers, and the average deliverable egg white protein formulation is 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).

Dosage of egg white protein formulations

The egg white protein formulations 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 suffering from egg allergy, and the dose is selected based on the stage of treatment and/or tolerance of the egg white protein. Briefly, patients orally ingest increasing amounts of egg white protein during an up-dosing phase, typically by administering an egg white protein formulation daily, with periodic dose escalation (e.g., increasing the dose once every two weeks), followed by a maintenance phase of higher dose levels. Further explanation of an exemplary oral immunotherapy dosing schedule is provided herein. For administration, the egg white protein formulation is removed from the dosage container and administered orally. For example, a capsule containing an egg white protein formulation is not entirely edible, but rather the egg white protein formulation is removed from the capsule prior to oral administration to a patient. Preferably, the egg white protein preparation is mixed with a food vehicle prior to consumption.

The amount of egg white protein preparation with a given egg white protein concentration packaged in a dosage container sets the amount of egg white protein (i.e. the dose) in the dosage container. Thus, the dosage of the container depends on the concentration of ovalbumin in the formulation and the amount of formulation added to the container. The expected dose of egg white protein in the dose container (i.e., label claim) may be in the range of, for example, about 0.1mg to about 600mg of egg white protein, or any amount within this range. By way of example, the dose (label statement) of the dose container may be 0.2mg, 1mg, 3mg, 6mg, 12mg, 20mg, 40mg, 80mg, 120mg, 160mg, 200mg, 240mg or 300mg of ovalbumin.

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

Because the dosage container itself is not ingested (the contents of the container are ingested), the material of the container need not be edible. However, in situations where the container is not intended to be consumed, it is useful to have an edible container. The container preferably limits exposure of the contents to moisture or air. Exemplary containers may be hypromellose-based containers (such as capsules) or foil-lined pouches. The container should be easy to open so that the egg white protein preparation in the container can flow out of the container.

In one example, an egg white protein formulation comprises, consists essentially of, or consists of: from about 0.1% to about 0.3% by weight of a dry egg white protein powder, from about 50% to about 70% by weight of a first diluent (such as pregelatinized starch), from about 35% to about 45% by weight of a second diluent (such as microcrystalline cellulose), and from 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 glidants 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 egg white protein. The egg white protein formulation may be packaged in a dosage container, such as a capsule, e.g., size 2 capsule, at an expected egg white protein dosage (i.e., label statement) of 0.2 mg. The dosage container may contain from about 170mg to about 190mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage vessel may be between about 0.15mg to about 0.25mg (such as about 0.17mg to about 0.23mg, about 0.18mg to about 0.22mg, or about 0.19mg to about 0.21 mg).

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

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

In another example, an egg white protein formulation comprises, consists essentially of, or consists of: about 0.5% to about 0.8% by weight of a 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 glidants or substantially free of colloidal silicon dioxide. In some embodiments, the egg white protein formulation comprises about 0.4% to about 0.64% egg white protein by weight. The egg white protein formulation may be packaged in a dosage container (such as a capsule, e.g., size 2 capsule) at an expected egg white protein dosage (i.e., label claim) of 1 mg. The dosage container may contain from about 170mg to about 190mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage vessel may be between about 0.75mg to about 1.25mg (such as about 0.85mg to about 1.15mg, about 0.9mg to about 1.1mg, or about 0.95mg to about 1.05 mg).

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

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

In another example, an egg white protein formulation comprises, consists essentially of, or consists of: about 4% to about 4.5% by weight of a 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 glidants or substantially free of colloidal silicon dioxide. In some embodiments, the egg white protein formulation comprises about 1.44% to about 1.92% egg white protein by weight. The egg white protein formulation may be packaged in a dosage container, such as a capsule, e.g., size 2 capsule, at an expected egg white protein dosage (i.e., label claim) of 6 mg. The dosage container may contain from about 170mg to about 190mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage vessel may be between about 4.5mg to about 7.5mg (such as about 5.1mg to about 6.9mg, about 5.4mg to about 6.6mg, about 5.7mg to about 6.3mg, or about 5.8mg to about 6.2 mg).

In another example, an 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 glidants or substantially free of colloidal silicon dioxide. In some embodiments, the egg white protein formulation comprises about 1.44% to about 1.92% egg white protein by weight. The egg white protein formulation may be packaged in a dosage container, such as a capsule, e.g., size 2 capsule, at an expected egg white protein dosage (i.e., label claim) of 6 mg. The dosage container may contain from about 170mg to about 190mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage vessel may be between about 4.5mg to about 7.5mg (such as about 5.1mg to about 6.9mg, about 5.4mg to about 6.6mg, about 5.7mg to about 6.3mg, or about 5.8mg to about 6.2 mg).

In another example, an egg white protein formulation comprises, consists essentially of, or consists of: about 2.5% to about 3.5% by weight of a 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 glidant (such as colloidal silicon dioxide). In some embodiments, the egg white protein formulation comprises about 2% to about 2.8% egg white protein by weight. The egg white protein formulation may be packaged in a dosage container, such as a capsule, e.g., a size 00 capsule, at an expected egg white protein dosage (i.e., label claim) of 12 mg. The dosage container may contain from about 480mg to about 520mg of the formulation. Because the actual fill level of the formulation may vary, the actual dose of egg white protein in the dosage vessel may be between about 9mg to about 15mg (such as about 10.2mg to about 13.8mg, about 10.8mg to about 13.2mg, about 11.4mg to about 12.6mg, or about 11.7mg to about 12.3 mg).

In another example, an 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 dioxide. In some embodiments, the egg white protein formulation comprises about 2% to about 2.8% egg white protein by weight. The egg white protein formulation may be packaged in a dosage container, such as a capsule, e.g., a size 00 capsule, at an expected egg white protein dosage (i.e., label claim) of 12 mg. The dosage container may contain from about 480mg to about 520mg of the formulation. Because the actual fill level of the formulation may vary, the actual dose of egg white protein in the dosage vessel may be between about 9mg to about 15mg (such as about 10.2mg to about 13.8mg, about 10.8mg to about 13.2mg, about 11.4mg to about 12.6mg, or about 11.7mg to about 12.3 mg).

In another example, an egg white protein formulation comprises, consists essentially of, or consists of: about 4.5% to about 5.5% by weight of a 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 glidant (such as colloidal silicon dioxide). In some embodiments, the egg white protein formulation comprises from about 3.6% to about 4.4% egg white protein by weight. The egg white protein formulation may be packaged in a dosage container, such as a capsule, e.g., a size 00 capsule, at an expected egg white protein dosage (i.e., label claim) of 20 mg. The dosage container may contain from about 480mg to about 520mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage vessel may be between about 15mg to about 25mg (such as about 17mg to about 23mg, about 18mg to about 22mg, about 19mg to about 21mg, or about 19.5mg to about 20.5 mg).

In another example, an 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 from about 3.6% to about 4.4% egg white protein by weight. The egg white protein formulation may be packaged in a dosage container, such as a capsule, e.g., a size 00 capsule, at an expected egg white protein dosage (i.e., label claim) of 20 mg. The dosage container may contain from about 480mg to about 520mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage vessel may be between about 15mg to about 25mg (such as about 17mg to about 23mg, about 18mg to about 22mg, about 19mg to about 21mg, or about 19.5mg to about 20.5 mg).

In another example, an egg white protein formulation comprises, consists essentially of, or consists of: about 8% to about 12% by weight of a 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 glidant (such as colloidal silicon dioxide). In some embodiments, the egg white protein formulation comprises about 6.4% to about 9.6% egg white protein by weight. The egg white protein formulation may be packaged in a dosage container, such as a capsule, e.g., a size 00 capsule, with an expected egg white protein dosage (i.e., label statement) of 40 mg. The dosage container may contain from about 480mg to about 520mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage vessel may be between about 30mg to about 50mg (such as about 34mg to about 46mg, about 36mg to about 44mg, about 38mg to about 42mg, or about 39mg to about 41 mg).

In another example, an 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% egg white protein by weight. The egg white protein formulation may be packaged in a dosage container, such as a capsule, e.g., a size 00 capsule, with an expected egg white protein dosage (i.e., label statement) of 40 mg. The dosage container may contain from about 480mg to about 520mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage vessel may be between about 30mg to about 50mg (such as about 34mg to about 46mg, about 36mg to about 44mg, about 38mg to about 42mg, or about 39mg to about 41 mg).

In another example, an egg white protein formulation comprises, consists essentially of, or consists of: about 16% to about 24% by weight of a 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 glidant (such as colloidal silicon dioxide). In some embodiments, the egg white protein formulation comprises about 12.8% to about 19.2% egg white protein by weight. The egg white protein formulation may be packaged in a dosage container, such as a capsule, e.g., a size 00 capsule, at an expected egg white protein dosage (i.e., label claim) of 80 mg. The dosage container may contain from about 480mg to about 520mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage vessel may be between about 60mg to about 100mg (such as about 68mg to about 92mg, about 72mg to about 88mg, about 76mg to about 84mg, or about 78mg to about 82 mg).

In another example, an 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% egg white protein by weight. The egg white protein formulation may be packaged in a dosage container, such as a capsule, e.g., a size 00 capsule, at an expected egg white protein dosage (i.e., label claim) of 80 mg. The dosage container may contain from about 480mg to about 520mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage vessel may be between about 60mg to about 100mg (such as about 68mg to about 92mg, about 72mg to about 88mg, about 76mg to about 84mg, or about 78mg to about 82 mg).

In another example, an egg white protein formulation comprises, consists essentially of, or consists of: about 24% to about 36% by weight of a 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 glidant (such as colloidal silicon dioxide). In some embodiments, the egg white protein formulation comprises about 19.2% to about 28.8% egg white protein by weight. The egg white protein formulation may be packaged in a dosage container, such as a capsule, e.g., a size 00 capsule, at an expected egg white protein dosage (i.e., label claim) of 120 mg. The dosage container may contain from about 480mg to about 520mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage vessel may be between about 90mg to about 150mg (such as about 102mg to about 138mg, about 108mg to about 132mg, about 114mg to about 126mg, or about 117mg to about 123 mg).

In another example, an 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% egg white protein by weight. The egg white protein formulation may be packaged in a dosage container, such as a capsule, e.g., a size 00 capsule, at an expected egg white protein dosage (i.e., label claim) of 120 mg. The dosage container may contain from about 480mg to about 520mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage vessel may be between about 90mg to about 150mg (such as about 102mg to about 138mg, about 108mg to about 132mg, about 114mg to about 126mg, or about 117mg to about 123 mg).

In another example, an egg white protein formulation comprises, consists essentially of, or consists of: about 32% to about 38% by weight of a 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 glidant (such as colloidal silicon dioxide). In some embodiments, the egg white protein formulation comprises about 25.6% to about 30.4% egg white protein by weight. The egg white protein formulation may be packaged in a dosage container, such as a capsule, e.g., a size 00 capsule, with an expected egg white protein dosage (i.e., label statement) of 160 mg. The dosage container may contain from about 480mg to about 520mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage vessel may be between about 120mg to about 200mg (such as about 136mg to about 184mg, about 144mg to about 176mg, about 152mg to about 168mg, or about 156mg to about 154 mg).

In another example, an 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% egg white protein by weight. The egg white protein formulation may be packaged in a dosage container, such as a capsule, e.g., a size 00 capsule, with an expected egg white protein dosage (i.e., label statement) of 160 mg. The dosage container may contain from about 480mg to about 520mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of egg white protein in the dosage vessel may be between about 120mg to about 200mg (such as about 136mg to about 184mg, about 144mg to about 176mg, about 152mg to about 168mg, or about 156mg to about 154 mg).

In another example, an egg white protein formulation comprises, consists essentially of, or consists of: about 40% to about 60% by weight of a 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 glidant (such as colloidal silicon dioxide). In some embodiments, the egg white protein formulation comprises about 32% to about 48% egg white protein by weight. The egg white protein formulation may be packaged in a dosage container, such as a capsule, e.g., a size 00 capsule, at an expected egg white protein dosage (i.e., label claim) of 200 mg. The dosage container may contain from about 480mg to about 520mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of ovalbumin in the dosage container may be between about 150mg to about 250mg (such as about 170mg to about 230mg, about 180mg to about 220mg, about 190mg to about 210mg, or about 195mg to about 205 mg).

In another example, an 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% egg white protein by weight. The egg white protein formulation may be packaged in a dosage container, such as a capsule, e.g., a size 00 capsule, at an expected egg white protein dosage (i.e., label claim) of 200 mg. The dosage container may contain from about 480mg to about 520mg of the formulation. Because the actual fill amount of the formulation may vary, the actual dose of ovalbumin in the dosage container may be between about 150mg to about 250mg (such as about 170mg to about 230mg, about 180mg to about 220mg, about 190mg to about 210mg, or about 195mg to about 205 mg).

In another example, an egg white protein formulation comprises, consists essentially of, or consists of: about 50% to about 70% by weight of a 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 glidant (such as colloidal silicon dioxide). In some embodiments, the egg white protein formulation comprises about 40% to about 56% by weight 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, with an expected egg white protein dosage (i.e., label statement) of 240 mg. The dosage container may contain from about 480mg to about 520mg 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 between about 180mg to about 300mg (such as about 204mg to about 276mg, about 216mg to about 264mg, about 228mg to about 252mg, or about 234mg to about 246 mg).

In another example, an 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 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, with an expected egg white protein dosage (i.e., label statement) of 240 mg. The dosage container may contain from about 480mg to about 520mg 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 between about 180mg to about 300mg (such as about 204mg to about 276mg, about 216mg to about 264mg, about 228mg to about 252mg, or about 234mg to about 246 mg).

In another example, an egg white protein formulation comprises, consists essentially of, or consists of: about 60% to about 85% by weight of a 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 glidant (such as colloidal silicon dioxide). In some embodiments, the egg white protein formulation comprises about 48% to about 68% egg white protein by weight. The egg white protein formulation may be packaged in a dosage container, such as a capsule, e.g., a size 00 capsule, at an expected egg white protein dosage (i.e., label claim) of 300 mg. The dosage container may contain from about 480mg to about 520mg 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 between about 225mg to about 375mg (such as about 255mg to about 345mg, about 270mg to about 330mg, about 285mg to about 315mg, or about 292mg to about 308 mg).

In another example, an 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% egg white protein by weight. The egg white protein formulation may be packaged in a dosage container, such as a capsule, e.g., a size 00 capsule, at an expected egg white protein dosage (i.e., label claim) of 300 mg. The dosage container may contain from about 480mg to about 520mg 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 between about 225mg to about 375mg (such as about 255mg to about 345mg, about 270mg to about 330mg, about 285mg to about 315mg, or about 292mg to about 308 mg).

In one aspect, the egg white protein preparation comprises the major egg white allergens Gal d 1, Gal d 2, Gal d 3 and Gal d 4. The amount of each individual allergen can be expressed as a percentage of total protein in the egg white protein preparation. In one example, a dose of an egg white protein preparation having a 300mg tag statement comprises about 300mg egg white protein and 78% Gal d 2, meaning that the dose comprises about 234mg Gal d 2. It is understood in the art that egg white proteins may be highly glycosylated. Thus, the mass of egg white allergen means the mass of 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 that the composition is suitable for oral immunotherapy of egg allergy.

In some embodiments, the egg white protein preparation comprises between about 5% and about 20% Gal d 1, such as any one of 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 d 1, as compared to total egg white protein mass.

In some embodiments, the egg white protein preparation comprises between about 45% to about 90% Gal d 2 compared to total egg white protein mass, such as any of 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 d 2.

In some embodiments, the egg white protein preparation comprises between about 1% and about 20% Gal d 3 compared to total egg white protein mass, such as any one of about 1% to about 5%, about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 5% to about 15% Gal d 3.

In some embodiments, the egg white protein preparation comprises between about 0.1% and about 10% Gal d 4 compared to total egg white protein mass, such as any one of about 0.1% to about 3%, about 3% to about 6%, about 6% to about 9%, about 7% to about 10% Gal d 4.

Process for preparing egg white protein formulations

The method of preparing the egg white protein formulation may affect the batch uniformity of the bulk formulation prepared, the content uniformity of the dose container batch, or the deliverability of the egg white protein formulation from the container. Because the dose of egg white protein administered during the course of oral immunotherapy can have a wide range (e.g., about 0.2mg to about 300mg), and dose uniformity is important to reduce adverse effects associated with treatment, formulation preparation methods for different doses are designed to produce dose containers with consistent doses.

The particular method of preparing the formulation may vary from dose to dose in order to produce consistent dosages. Process development efforts to optimize blend uniformity and content uniformity are focused on lower doses (e.g., doses less than about 3 mg) and process development efforts to ensure adequate formulation flowability are focused on higher doses (e.g., about 300 mg). Methods have also been developed for moderate dosage strengths to adequately balance the desired properties of the final product.

The methods described herein also overcome challenges associated with scaling up of the manufacturing process. Ensuring lot uniformity with large quantities of formulations can be challenging, and the methods described herein overcome these challenges. The method of preparation may be used even when preparing batches of egg white protein formulation of about 5kg or more, such as from about 5kg to about 100kg, for example from about 5kg to about 7kg, from about 7kg to about 10kg, from about 10kg to about 15kg, from about 15kg to about 25kg, from about 25kg to about 50kg or from about 50kg to about 100 kg.

Preparation steps such as mixing (e.g., using a drum blender or cone mill), passing one or more powders or mixtures through a mesh screen and/or serial dilution when a diluent is added to the mixture are used during the preparation process. The specific steps used may depend on the egg white protein concentration in the formulation or the labeling statement for the dosage form (i.e., the expected amount of egg white protein formulation for the dosage form). Generally, one or more higher shear mixing steps (higher shear relative to other steps in the manufacturing process) are incorporated into the manufacturing process sequence to uniformly disperse the dry egg white protein powder into the mixture. The higher shear mixing also disperses soft low density agglomerates of colloidal silica (if present in the formulation), which are typically observed in colloidal silica feedstocks. The use of higher force mixing is not intended to reduce the primary particle size of any of the mixture components.

After the formulation has been prepared, the quality of one or more allergenic egg white proteins (e.g., egg white protein, ovomucoid, ovotransferrin, and/or lysozyme) in the formulation and/or the blend uniformity of the formulation may be evaluated. The quality of the one or more allergenic proteins can be assessed, for example, by characterizing one or more of ovalbumin, ovomucoid, ovotransferrin, and/or lysozyme (e.g., by determining the amount or relative amount of the one or more proteins, or the potency or relative potency of the one or more proteins). Blend uniformity of a formulation can be assessed by determining the protein content of the formulation, which can be compared to an expected protein content (e.g., label claim or expected concentration) of the formulation. For example, determining blend uniformity of a formulation can include identifying an expected protein content associated with the formulation, measuring the protein content in a sample from the formulation, and comparing the measured protein content to the expected protein content. Multiple samples from the formulation (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more samples) can be used to measure blend uniformity, and the average blend uniformity and/or Relative Standard Deviation (RSD) can be measured. By using the preparation methods described herein, the blend uniformity RSD from multiple samples of 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%).

The prepared formulation may 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 that occur during preparation and packaging, there may be some deviation from the label statement and the actual amount of egg white protein in a single dose container. Thus, the content uniformity of the prepared bulk dose containers can be measured. Content uniformity is based on the deliverable protein content of the formulation from the dosage container. The dosage container is associated with an expected amount of egg white protein in the dosage container. Thus, determining content uniformity for a plurality of dosage containers can include identifying an expected protein content associated with the plurality of dosage containers, measuring a deliverable protein content of a sample (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more dosage containers) taken from the plurality of dosage containers, and comparing the measured protein content to the expected protein content. When multiple samples are taken from multiple dose containers, the average content uniformity and/or content uniformity RSD can be determined. By using the manufacturing methods described herein, the content uniformity RSD of the plurality of dose containers is about 12% or less (e.g., about 4% to about 12%, or about 5% to about 12%).

Low dose manufacturing process

Blend uniformity and content uniformity are generally considered to be at greater risk of low dose strengths corresponding to low percentages of drug substance in the blend. These lower doses in the dosage vessel are typically about 12mg egg white protein or less, but a similar method can be used to produce larger doses. The formulation preparation methods typically produce a formulation having about 0.05 wt.% to about 4 wt.% egg white protein (such as between about 0.1 wt.% to about 0.7 wt.%, about 0.7 wt.% to about 1.5 wt.%, about 1.5 wt.% to about 2.5 wt.%, or about 2.5 wt.% to about 4 wt.% egg white protein).

Egg white protein formulations are typically prepared comprising two diluents (e.g., pregelatinized starch and microcrystalline cellulose). One or more diluents are mixed with the egg white protein powder in a stepwise manner (i.e., serial dilution of the egg white protein powder), wherein the egg white protein powder and a portion (but not all) of the diluent are mixed prior to adding an additional amount of diluent to the mixture. Passing the first mixture comprising the egg white protein powder and the first portion of diluent through a mesh screen prior to further serial dilution enhances blend uniformity of the fully prepared egg white protein formulation.

As shown in fig. 1, the dry egg white protein powder is mixed with a first amount of a first diluent (e.g., pregelatinized starch) at step 102. The dried egg white protein powder and the first amount of the first diluent may be added to any suitable container, such as a tray or bag, and may be mixed together before proceeding to the next step. Mixing may be performed, for example, by shaking the container, or by using an impeller, blender (such as a drum blender), or any other suitable device.

Once the dry egg white protein powder is mixed with a first amount of a first diluent to form a first mixture, the first mixture is passed through a mesh screen (see step 104 of fig. 1). The mesh screen typically has a size of about 250 μm to about 850 μm (such as 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.

Once the first mixture has passed through the mesh screen, the mixture may 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 may be co-screened 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, blender (such as a drum blender), or any other suitable device. Additional amounts of the first diluent may be added in a stepwise manner to serially dilute the mixture, as indicated by arrow 108 in fig. 1. For example, the first mixture may be mixed with the second amount of the first diluent in a plurality of iterative sub-steps. The substeps may comprise adding a portion of the second amount of the first diluent to the first mixture and mixing the portion of the second amount of the first diluent with the first mixture. Another portion of the second amount of the first diluent may then be added and further mixed. These dilution substeps may be performed 1, 2, 3, 4, 5 or more times until the desired amount of the first diluent has been mixed with the dry egg white protein powder. Optionally, the mixture may be passed through a mesh screen before adding an additional portion of the second amount of the first diluent.

Alternatively, the first mixture and the second amount of the first diluent may be mixed together by continuously mixing the first mixture while the second amount of the first diluent is added to the mixture. That is, rather than gradually adding the second amount of the first diluent to the first mixture to form the second mixture, the mixture is mixed while the second amount of the first diluent is added.

Once the second mixture is formed, the second mixture may 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 may be mixed by shaking the combined components, or by using an impeller, blender (such as a drum 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 at step 112. The higher shear mixing may occur after the initial mixing of the second mixture with the second diluent or may be the mixing force used to mix the second mixture with the second diluent. This can be done, for example, using a cone mill (which can be equipped, for example, using a circular 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 initially form a third mixture, and in some embodiments, the second mixture and the second diluent are premixed to form the third mixture before the third mixture is subjected to the higher shear mixing. The higher shear mixing increases the uniform dispersion of the dried egg white protein powder in the formulation and disperses soft, low density agglomerates that may form. Mixing using higher shear forces is not intended to reduce the primary particle size of any mixture components, but rather is merely intended to dissociate the agglomerates of particles.

A lubricant (e.g., magnesium stearate) is added to the mixture to form an egg white protein formulation, as shown in step 114. The lubricant may be added to any mixture during the manufacturing process, but at some point is mixed with a mixture comprising the dried egg white protein powder, the first diluent, and the second diluent. For example, the lubricant may be mixed with the third mixture before, after, or during the higher shear mixing step. Additional amounts of one or more diluents (e.g., a first diluent, such as pregelatinized starch) can be co-added to the mixture with the lubricant. Additionally, 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, the lubricant and an additional amount of the first diluent are mixed (and optionally, the mixture is passed through a mesh screen) prior to mixing with the egg white protein-containing mixture.

Optionally, the egg white protein preparation may be passed through a mesh screen. The mesh screen typically has a size of about 250 μm to about 850 μm (such as about 300 μm to about 710 μm, or about 425 μm to about 600 μm).

Blend uniformity or quality characteristics of a batch of egg white protein formulations produced using these methods can be determined, as further described herein. In some embodiments, the method further comprises determining blend uniformity and/or quality characteristics of the egg white protein formulation.

In an exemplary method of preparing an egg white protein formulation, the method may comprise (a) mixing a dry egg white protein powder with a first amount of a first diluent (such as pregelatinized starch) to form a first mixture; (b) co-screening 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 a 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; (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 the first part of the sixth mixture with a lubricant (such as magnesium stearate) and optionally a sixth part of the first diluent to form a seventh mixture; (j) passing the seventh mixture through a mesh screen; and (k) mixing the screening mixture of (j) with a second portion of the sixth mixture to form an egg white protein preparation. Step (g) may comprise two mixing sub-steps, wherein one mixing sub-step is at a higher shear force than the other mixing sub-step. In an exemplary embodiment, step (g) optionally includes two substeps, wherein the first substep is at a lower shear force than the second substep (such as with a drum blender), and the second substep is at a higher shear force than the first substep (such as with a cone mill). This method of preparing a formulation is particularly useful for preparing formulations containing lower dose dosage containers of the formulation, such as dosages of from about 0.1mg to about 12mg (such as dosage containers of about 0.2mg, about 1mg, about 3mg, about 6mg or about 12mg, or any dosage therebetween). The formulation may have, for example, about 0.05% to about 2.5% egg white protein by weight. In some embodiments, the formulation is free or substantially free of glidants. In some embodiments, the formulation is free or substantially free of colloidal silica. Blend uniformity or quality characteristics of a batch of egg white protein formulations produced using these methods can be determined, as further described herein. In some embodiments, the method further comprises determining blend uniformity and/or quality characteristics of the egg white protein formulation.

An exemplary method of preparing a low dose formulation of ovalbumin is shown in fig. 12A. In step 402, a dry egg white protein powder (which is preferably characterized by ensuring total protein levels and specific allergen levels) 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 dried egg white protein powder prior to mixing with the first amount of the first diluent. In step 404, the first mixture is co-sieved with a second amount of the first diluent. In step 406, the co-sifted first mixture and a second amount of the first diluent are mixed to form a second mixture. Step 408 allows for the addition of additional amounts of the first diluent to the second mixture in a stepwise manner. Step 408 may include one, two, three, or more substeps of adding additional amounts of the first diluent and mixing. Performing these steps continuously allows 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 (such as by a cone mill) to disperse the particle aggregates. 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 the 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 an egg white protein preparation. Blend uniformity or quality characteristics of a batch of egg white protein formulations produced using these methods can be determined, as further described herein. In some embodiments, the method further comprises determining blend uniformity and/or quality characteristics of the egg white protein formulation.

In another exemplary method of preparing an egg white protein formulation, the method may comprise (a) mixing a dry egg white protein powder with a first amount of a first diluent (such as pregelatinized starch) to form a first mixture; (b) co-screening the first mixture with a second portion of the first diluent through a mesh screen; (c) mixing the first mixture with a 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; (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) optionally mixing the sixth mixture using higher shear forces than the shear forces used to mix the fifth mixture with the second diluent in step (g); (i) mixing a lubricant (such as magnesium stearate) with the sixth portion of the first diluent to form a seventh mixture, and optionally passing the seventh mixture through a mesh screen; and (k) mixing the seventh mixture with the sixth mixture to form an egg white protein preparation. This method of preparing a formulation is particularly useful for preparing formulations containing lower dose dosage containers of the formulation, such as dosages of from about 0.1mg to about 12mg (such as dosage containers of about 0.2mg, about 1mg, about 3mg, about 6mg or about 12mg, or any dosage therebetween). The formulation may have, for example, about 0.05% to about 2.5% egg white protein by weight. In some embodiments, the formulation is free or substantially free of glidants. In some embodiments, the formulation is free or substantially free of colloidal silica. In some embodiments, blend uniformity or quality characteristics of the dried egg white protein powder (i.e., prior to formulation) and/or egg white protein preparation (i.e., after formulation) produced using these methods can be determined, as further described herein, such as using HPLC assays (e.g., RP-HPLC) or ELISA assays to determine the concentration or profile of one or more of egg white protein, 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.

An exemplary method of preparing a low dose formulation of ovalbumin is shown in fig. 12B. At step 422, the dried egg white protein powder is mixed with a first portion of a first diluent (such as pregelatinized starch) to form a first mixture. At step 424, the first mixture is co-screened through a mesh screen with a second portion of the first diluent, and the combined first mixture and second portion of the first diluent are mixed to form a second mixture at step 426. At step 428, the second mixture is mixed (e.g., using a blender, such as a drum blender) with a third portion of the first diluent to form a third mixture. At step 430, the third mixture is mixed (e.g., using a blender, such as a drum blender) with a fourth portion of the first diluent to form a fourth mixture. At step 432, the fourth mixture is mixed (e.g., using a blender, such as a drum blender) with a fifth portion of the first diluent to form a fifth mixture. At 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 higher shear forces than those used at step 434, for example using a cone mill. At 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 at step 438 to form an eighth mixture (e.g., a formulated composition).

In another exemplary method of preparing an egg white protein formulation, the method may comprise (a) mixing a dry egg white protein powder with a first amount of a first diluent (such as pregelatinized starch) to form a first mixture; (b) co-sifting the first mixture with a second portion of the first diluent through a mesh screen, and optionally mixing the co-sifted composition; (c) serially diluting the co-sieved composition with one or more additional portions of the first diluent (e.g., 1, 2, 3, 4, 5, or more additional portions) and mixing the composition after adding each 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 another 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 preparing a formulation is particularly useful for preparing formulations containing lower dose dosage containers of the formulation, such as dosages of from about 0.1mg to about 12mg (such as dosage containers of about 0.2mg, about 1mg, about 3mg, about 6mg or about 12mg, or any dosage therebetween). The formulation may have, for example, about 0.05% to about 2.5% egg white protein by weight. In some embodiments, the formulation is free or substantially free of glidants. In some embodiments, the formulation is free or substantially free of colloidal silica. In some embodiments, blend uniformity or quality characteristics of the dried egg white protein powder (i.e., prior to formulation) and/or egg white protein preparation (i.e., after formulation) produced using these methods can be determined, as further described herein, such as using HPLC assays (e.g., RP-HPLC) or ELISA assays to determine the concentration or profile of one or more of egg white protein, 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.

An exemplary method of preparing a low dose formulation of ovalbumin is shown in fig. 12C. At step 440, the dry egg white protein powder is mixed with a first portion of a first diluent (such as pregelatinized starch) to form a first mixture. At step 442, the first mixture is co-screened with the second portion of the first diluent through a mesh screen. At step 444, the first mixture is mixed with a second amount of a first diluent to form a second mixture. At 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. Serial dilution may be performed, for example, in 1, 2, 3, 4, 5, or more substeps, wherein a portion of the first diluent is mixed with the composition at each substep, and then additional portions of the first diluent are added. At step 448, the third mixture is mixed with a second diluent (such as microcrystalline cellulose) to form a fourth mixture. At 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 at step 452 to form a formulated composition.

The egg white protein formulation may then be packaged in a package, such as a capsule or sachet, to obtain the desired dose of egg white protein in a dosage container. The dosage container may contain from about 100mg to about 1000mg (such as from about 100mg to about 250mg, from about 250mg to about 400mg, from about 400mg to about 600mg, or from about 600mg to about 1000mg) of the egg white protein formulation. By way of example, in some embodiments, about 180mg of an egg white protein formulation is contained in a container (such as a capsule) to obtain a desired amount of egg white protein formulation in a dosage container.

Once the egg white protein formulation is packaged in a dosage container, the content uniformity of the dosage container can be determined.

Intermediate dosage preparation method

Blend uniformity and content uniformity are balanced with the flowability of the egg white protein formulation when preparing the formulation for a particular dose, such as about 3mg to about 300mg in a dosage container, although similar methods can be used to produce larger doses. The formulation preparation methods generally produce formulations having about 1.5 wt.% to about 60 wt.% egg white protein (such as between about 1.5 wt.% to about 3 wt.%, about 3 wt.% to about 8 wt.%, about 8 wt.% to about 15 wt.%, about 15 wt.% to about 30 wt.%, or about 30 wt.% to about 60 wt.% egg white protein).

The egg white protein formulations prepared typically comprise two diluents (e.g. pregelatinized starch and microcrystalline cellulose), a lubricant and optionally a glidant (such as colloidal silicon dioxide). However, for some medium strength dosage forms, colloidal silica is not included in the formulation.

As shown in fig. 2, at step 202, a dry 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 glidant (such as colloidal silicon dioxide) may also be mixed with the dried egg white protein powder at this step. Mixing is preferably accomplished using higher shear forces to disperse the particle aggregates. This can be done, for example, using a cone mill mixer (which can be configured with either a round mill impeller or a square mill impeller).

The first mixture may 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 fig. 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, blender (such as a drum blender), or any other suitable device.

The second mixture may be mixed with a second diluent (e.g., microcrystalline cellulose) to form a third mixture, as shown in step 206 of fig. 2. Optionally, an additional amount of the first diluent is added to the second mixture or the third mixture. The second mixture may be mixed with an additional amount of the first diluent prior to mixing with the second diluent. Alternatively, additional amounts of the first diluent may be co-mixed with the second mixture and the second diluent. The second mixture and the second diluent (and optionally additional amounts of the first diluent) can be mixed by shaking the combined components, or by using an impeller, blender (such as a drum blender), or any other suitable device.

The third mixture is also subjected to mixing at a higher shear force than that used to form the second mixture, as shown in step 208, which can disperse the particle aggregates in the mixture. The higher shear mixing may occur after the initial mixing of the second mixture with the second diluent or may be the mixing force used to mix the second mixture with the second diluent. This may be done, for example, using a cone mill (which may be equipped, for example, using a circular 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 initially form a third mixture, and in some embodiments, the second mixture and the second diluent are premixed to form the third mixture before the third mixture is subjected to the higher shear mixing. The higher shear mixing increases the uniform dispersion of the dried egg white protein powder in the formulation and disperses soft, low density agglomerates that may form. Mixing using higher shear forces is not intended to reduce the primary particle size of any mixture components, but rather is merely intended to dissociate the agglomerates of particles.

A lubricant (e.g., magnesium stearate) is added to the mixture to form an egg white protein formulation, as shown in step 210. The lubricant may be added to any mixture during the manufacturing process, but at some point is mixed with a mixture comprising the dried egg white protein powder, the first diluent, and the second diluent. For example, the lubricant may be mixed with the third mixture before, after, or during the higher shear mixing step. Additional amounts of one or more diluents (e.g., a first diluent, such as pregelatinized starch) can 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.

An exemplary method of preparing a medium dosage formulation of ovalbumin is shown in fig. 13A. In step 502, a dry egg white protein powder (which is preferably characterized by ensuring total protein levels and specific allergen levels) 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 glidant (such as colloidal silicon dioxide) to improve flowability. In some embodiments, step 502 further comprises characterizing the dried egg white protein powder prior to mixing with the first amount of the first diluent. In step 504, the first mixture is mixed with a second amount of a first diluent to form a second mixture. The mixing step of 504 may include two sub-steps, one of which is at a higher shear force than the other. In an exemplary embodiment of the method, step 504 includes two substeps, where the first substep is at a higher shear force (such as with a cone mill) than the second substep, and the second substep is at a lower shear force (such as with a drum blender). Step 506 is optional and allows for the addition of additional amounts 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 using a high shear mixing step (such as a cone mill) to disperse the particle aggregates. 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 mixed with an additional amount of the dispersed third mixture, followed by mixing in step 518 to form an egg white protein preparation.

In some methods of making an egg white protein preparation, the method comprises (a) mixing a 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 a second diluent to form a fourth mixture; (e) mixing the fourth mixture with a fourth portion of the first diluent and the lubricant to form a fifth mixture; (f) passing the fifth mixture through a mesh screen; (g) mixing the screening mixture of (f) with an additional amount of the fourth mixture to form an egg white protein preparation. In step (b), the mixing step may comprise two sub-steps, one of which is at a higher shear force than the other. In an exemplary embodiment, step (b) comprises two sub-steps, wherein the first sub-step comprises mixing with a higher shear force than the second sub-step (such as with a cone mill), and the second sub-step comprises mixing with a blender (such as with a drum blender). In step (d), the mixing step may comprise two sub-steps, one of which is at a higher shear force than the other. In one exemplary embodiment, step (d) comprises two sub-steps, wherein the first sub-step comprises mixing with lower shear forces than the second sub-step. In some embodiments, the formulation is free or substantially free of glidants. In some embodiments, the formulation is free or substantially free of colloidal silica. In some embodiments, blend uniformity or quality characteristics of the dried egg white protein powder (i.e., prior to formulation) and/or egg white protein preparation (i.e., after formulation) produced using these methods can be determined, as further described herein, such as using HPLC assays (e.g., RP-HPLC) or ELISA assays to determine the concentration or profile of one or more of egg white protein, 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.

In another method of preparing an egg white protein formulation, the method comprises (a) mixing a 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, and optionally mixing the second mixture with lower shear forces than the shear forces used to mix the first mixture with the second portion of the first diluent; (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, and optionally mixing the fourth mixture using higher shear forces than those used to mix the third mixture with the second diluent; (e) mixing the fourth portion of the first diluent with a lubricant (such as magnesium stearate) to form a fifth mixture, and 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 glidants. In some embodiments, the formulation is free or substantially free of colloidal silica. In some embodiments, blend uniformity or quality characteristics of the dried egg white protein powder (i.e., prior to formulation) and/or egg white protein preparation (i.e., after formulation) produced using these methods can be determined, as further described herein, such as using HPLC assays (e.g., RP-HPLC) or ELISA assays to determine the concentration or profile of one or more of egg white protein, 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.

Figure 13B illustrates another exemplary method of preparing an egg white protein formulation. Step 520 includes mixing the dry egg white protein powder with a first portion of a first diluent (such as pregelatinized starch) to form a first mixture. At step 522, 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 can be mixed with the second portion of the first diluent using a first mixing shear force (e.g., using a cone mill) to form a second mixture, and the second mixture can be further mixed using a second shear force that is lower than the first shear force (e.g., using a blender, such as a drum blender). At step 524, the second mixture is mixed with a third portion of the first diluent to form a third mixture. At 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 can be mixed with the second diluent using a first mixing shear force (e.g., using a blender, such as a drum blender) to form a fourth mixture, and the fourth mixture can be further mixed using a second shear force that is higher than the first shear force (e.g., using a cone mill). At step 528, the 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. At step 530, the fourth mixture is mixed with the fifth mixture to form an egg white protein preparation.

In another method of preparing an egg white protein formulation, the method comprises (a) mixing a dry egg white protein powder with a first amount of a first diluent (such as pregelatinized starch) and a glidant (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, and optionally mixing the second mixture with lower shear forces than the shear forces used to mix the first mixture with the second portion of the first diluent; (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, and optionally mixing the fourth mixture using higher shear forces than the shear forces 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, and optionally passing the fifth mixture through a mesh screen; and (f) mixing the fourth mixture with the fifth mixture. In some embodiments, blend uniformity or quality characteristics of the dried egg white protein powder (i.e., prior to formulation) and/or egg white protein preparation (i.e., after formulation) produced using these methods can be determined, as further described herein, such as using HPLC assays (e.g., RP-HPLC) or ELISA assays to determine the concentration or profile of one or more of egg white protein, 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.

Figure 13C illustrates another exemplary method of preparing 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 glidant (such as colloidal silicon dioxide) to form a first mixture. At 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 can be mixed with the second portion of the first diluent using a first mixing shear force (e.g., using a cone mill) to form a second mixture, and the second mixture can be further mixed using a second shear force that is lower than the first shear force (e.g., using a blender, such as a drum blender). At 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 can be mixed with the second diluent using a first mixing shear force (e.g., using a blender, such as a drum blender) to form a fourth mixture, and the fourth mixture can be further mixed using a second shear force that is higher than the first shear force (e.g., using a cone mill). At step 538, the 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. At step 540, the fifth mixture is mixed with the fourth mixture.

Once the egg white protein formulation is packaged in a dosage container, the content uniformity of the dosage container can be determined.

High dose manufacturing process

The preparation of egg white protein formulations for certain strength doses, such as about 100mg or higher, is designed to prioritize the flowability of the formulation. The egg white protein powder may adhere to the packaging material, thereby limiting the deliverability of the formulation from the container. To increase flowability, formulations are typically prepared with glidants, 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 from about 50% to about 80% (such as from about 50% to about 60%, from about 60% to about 70%, or from about 70% to about 80%) by weight egg white protein.

To prepare an egg white protein formulation, a dry egg white protein powder, a first diluent (such as pregelatinized starch), and a glidant (such as colloidal silicon dioxide) are mixed together, as shown in step 302 of fig. 3. The components may be mixed by shaking the combined components, or by using an impeller, blender (such as a drum blender), or any other suitable device.

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 may be mixed using a higher shear force than the shear force used to mix the egg white protein powder with the first diluent and the glidant. Higher shear forces can dissociate particle agglomerates within the mixture.

The second mixture is also mixed with a lubricant to form an egg white protein formulation, as shown in step 306. Optionally, additional amounts of the first diluent may also be mixed with the second mixture before, after, or simultaneously with the mixing of the lubricant 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, 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 second mixture to form the egg white protein formulation.

An exemplary method of preparing a high dose formulation of ovalbumin is shown in figure 14A. In step 602, a dry egg white protein powder (which is preferably characterized by ensuring total protein levels and specific allergen levels) is mixed with a first amount of a first diluent (such as pregelatinized starch) and a glidant (such as colloidal silicon dioxide) to form a first mixture. In some embodiments, step 602 further comprises characterizing the dried egg white protein powder prior to 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 cone mill) to disperse the particle aggregates. 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 an egg white protein preparation.

In some methods of making an egg white protein formulation, the method comprises (a) mixing a 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 than the shear 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, and optionally passing the third mixture through a mesh screen; and (d) mixing the second mixture with the third mixture. In some embodiments, blend uniformity or quality characteristics of the dried egg white protein powder (i.e., prior to formulation) and/or egg white protein preparation (i.e., after formulation) produced using these methods can be determined, as further described herein, such as using HPLC assays (e.g., RP-HPLC) or ELISA assays to determine the concentration or profile of one or more of egg white protein, 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.

Another exemplary method of preparing a high dose formulation of ovalbumin is shown in figure 14B. At step 614, the dried egg white protein powder and a first portion of a first diluent (such as pregelatinized starch) are mixed with a glidant (such as colloidal silicon dioxide) to form a first mixture. At step 616, the first mixture is mixed with a second diluent (such as microcrystalline cellulose) to form a second mixture. In some embodiments, the first mixture is mixed with a second diluent (e.g., using a cone mill) at a higher shear force than the dried egg white protein powder is mixed with the first portion of the first diluent and the glidant at step 614 to form the first mixture (which may be mixed, for example, using a blender such as a drum blender). At step 618, the 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. At step 620, the second mixture is mixed with the third mixture.

The egg white protein formulation may then be packaged in a package, such as a capsule or sachet, to obtain the desired dose of egg white protein in a dosage container. The dosage container may contain from about 100mg to about 1000mg (such as from about 100mg to about 250mg, from about 250mg to about 400mg, from about 400mg to about 600mg, or from about 600mg to about 1000mg) of the egg white protein formulation. By way of example, in some embodiments, about 500mg of an egg white protein formulation is contained in a container (such as a capsule) to obtain a desired amount of egg white protein formulation in a dosage container.

Once the egg white protein formulation is packaged in a dosage container, the content uniformity of the dosage container can be determined.

Quality control method

The dosage forms and egg white protein formulations described herein are useful for oral immunotherapy, wherein an increased dose is administered to a patient allergic to one or more egg proteins to desensitize the patient to these proteins. Since the allergic response to allergenic proteins may be severe and even life-threatening, it is desirable to have both sufficient quantitative and qualitative control over the administered dose. To ensure quality control of the product administered to the patient, various parameters of the dry egg white protein powder, the egg white protein formulation, and the dosage container (and dosage container batch) containing the egg white protein formulation may be monitored. Such quality control methods may include characterizing one or more allergenic egg white proteins (ovomucoid, egg white, ovotransferrin, and/or lysozyme), determining protein content, determining blend uniformity of an egg white protein formulation, determining content uniformity or deliverable mass of bulk dose containers comprising an egg white protein formulation, or determining water activity of a dry egg white protein powder or an egg white protein formulation. Quality control methods may be included in the manufacturing process to ensure consistent manufacture of the egg white protein formulation or dosage container.

Allergenic proteins in egg white include Gal d 1 (ovomucoid), Gal d 2 (ovalbumin), Gal d 3 (ovotransferrin), and Gal d 4 (lysozyme). One or more (or all) of these proteins in an egg white protein formulation or a dried egg white protein powder can be characterized using the methods described herein. Characterization of the one or more allergenic egg white proteins can include determining the presence of the one or more proteins, the amount or relative amount of the one or more immunodominant egg white proteins, or the potency or relative potency of the one or more immunodominant egg white proteins.

One method of characterizing ovomucoid, ovalbumin, ovotransferrin and/or lysozyme includes analyzing dry ovalbumin powder or ovalbumin formulations using High Performance Liquid Chromatography (HPLC). Exemplary HPLC techniques include size exclusion chromatography (SEC-HPLC) and reverse phase HPLC (RP-HPLC). For example, RP-HPLC separates all four allergenic egg white proteins well for qualitative or quantitative analysis. HPLC profiles can be obtained and peaks representing ovomucoid, ovalbumin, ovotransferrin and/or lysozyme can be identified or compared to reference standards for qualitative assessment.

Characterizing the ovomucoid, ovalbumin, ovotransferrin and/or lysozyme may comprise determining the presence of one or more of the ovomucoid, ovalbumin, ovotransferrin and/or lysozyme in a dry ovalbumin powder (drug substance) and/or an ovalbumin formulation (drug product). This can be done, for example, by polyacrylamide gel electrophoresis (PAGE), such as SDS-PAGE, immunoblotting or HPLC, such as SEC-HPLC or RP-HPLC. Allergenic proteins are typically eluted from the HPLC column at consistent time points, and peaks can be identified using reference standards such as purified commercially available ovomucoid, ovalbumin, ovotransferrin, or lysozyme. The allergenic proteins in the dried egg white protein powder or egg white protein preparation can be identified using HPLC profiles, e.g., based on retention time. Qualitative comparisons of the HPLC profile to reference standards can also be made to determine the identity of the tested dried egg white protein powder or egg white protein formulation.

Characterizing the ovomucoid, ovalbumin, ovotransferrin, and/or lysozyme may include quantifying the amount of the ovomucoid, ovalbumin, ovotransferrin, and/or lysozyme in an ovalbumin protein formulation or a dried ovalbumin protein powder. HPLC (e.g., SEC-HPLC or RP-HPLC) methods can be used to fully quantify the protein, for example, by determining peak areas in the HPLC profile. The concentration of ovomucoid, ovalbumin, ovotransferrin and/or lysozyme in an egg white protein formulation or a dried egg white protein powder can be determined, for example, using the method by comparing protein peaks associated with ovomucoid, ovalbumin, ovotransferrin and/or lysozyme to a reference. Another example of characterizing proteins includes determining the relative amounts of ovomucoid, ovalbumin, ovotransferrin, and/or lysozyme, which can be determined using HPLC profiling. The relative amount may be compared to the total protein in the egg white protein preparation or in the dried egg white protein preparation (e.g., by comparison 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 the protein as it elutes from the column, such as wavelengths of light from about 210nm to about 280 nm. The peak area due to the protein in the HPLC profile correlates with the amount of the protein in the measurement sample. However, the peak area ratio between different proteins may not necessarily reflect the weight ratio of the different proteins due to the difference in absorbance of the proteins. The weight ratio of the different proteins can be determined by calibrating the peak area to the mass of the protein under study. Thus, the relative amount of protein may be, for example, the relative peak area from an HPLC profile or the relative weight of the protein. Since quality control methods are used to ensure consistency between batches, any basis for the relative amounts of allergenic proteins may be used.

One or more of the ovomucoid, ovalbumin, ovotransferrin, and/or lysozyme may also be characterized to determine the potency or relative potency (relative to the potency of the protein in a reference sample) of the allergenic ovalbumin protein in the dried ovalbumin protein powder or ovalbumin protein formulation. The potency or relative potency of the allergenic protein can change due to denaturation of the allergenic protein, and measuring the potency or relative potency of the allergenic protein in a dried egg white protein powder or egg white protein formulation can be used 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 a dried egg white protein powder or egg white protein formulation is measured in vitro. In some embodiments, a single allergenic protein (e.g., an ovomucoid) is used to express the potency or relative potency of a powder or formulation. Potency can be reported, for example, as EC measured by potency assay₅₀Or EC₅₀Ratio (i.e., relative potency). Exemplary immunoassay techniques that may be used to determine the potency of one or more allergenic egg white proteins include enzyme linked immunosorbent assays (ELISAs), Radioimmunoassays (RIA), immunoblots, Surface Plasmon Resonance (SPR), or multiplex immunoassays. Measurement of egg white Another method of potency of ovalbumin, ovotransferrin, ovomucoid, or lysozyme in a protein formulation may include cell-based assays (e.g., basophil histamine release assays). See, for example, Santos et al, basic activation test, food exchange in a test tube or specialized research tool? Clinical and Translational Allergy, volume 6, No. 10, pages 1-9 (2016).

The reporter molecules used in the potency assay bind the assayed allergenic proteins with high specificity and are typically antibodies. The antibody may be a monoclonal antibody or a polyclonal antibody. In some embodiments, the antibody is an IgG antibody or an IgE antibody. Antibodies used to determine potency or relative potency specifically bind to the protein being tested (e.g., ovalbumin, ovotransferrin, ovomucoid, or lysozyme). Antibody libraries comprising antibodies that specifically bind two or more different proteins can also be used to determine the potency or relative potency of two or more assay proteins simultaneously. For example, the library can include two or more of an antibody that specifically binds to ovalbumin, an antibody that specifically binds to ovotransferrin, an antibody that specifically binds to an ovomucoid, and/or an antibody that specifically binds to lysozyme. The antibody library may be a separately purified antibody library mixed together or may be derived from an animal immunized with whole egg or egg white protein. When measuring relative potency, the same antibody or antibody library should be used for the test sample and the reference sample.

The protein content of the dried egg white protein powder or egg white protein formulation can be determined before or after packaging into a dosage form to monitor quality during the manufacturing process. Protein content may be reported as a concentration (e.g., weight percent of the composition) or a target (i.e., expected) amount of a reference protein (e.g., X% of the target dose). The protein content of the dried egg white protein powder is typically measured to determine how much dried egg white protein powder should be included in the manufacturing process to achieve the desired egg white protein concentration in the prepared egg white protein formulation. Protein content may also be used to measure blending uniformity in an egg white protein formulation or content uniformity in a bulk prepared dosage container containing an egg white protein formulation. Exemplary methods of measuring protein content include absorbance, Lowry assay, Bradford assay, combustion assay, bicinchoninic acid (BCA) assay, HPLC (such as SEC-HPLC, RP-HPLC, or any other chromatography method that can quantitatively measure the amount of protein), or any other suitable quantitative protein assay.

The deliverable mass of an egg white protein preparation from a dosage container can be measured on an individual dosage container basis or as a sample taken from a preparation lot of dosage containers. The deliverable mass of a batch of dose containers can be determined by sampling a plurality of dose containers (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more dose containers) selected from the batch of dose containers. The composition can be delivered from the container when it can be recovered 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 amount of dosage form that can be delivered from the dosage container under normal use. Compositions that can only be removed from a container by inserting a mechanical device into the container or adding a fluid (such as a compressed gas or liquid) to the container cannot be delivered from the container.

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

Oral immunotherapy method

The patients treated by using the oral immunotherapy methods described herein are human individuals suffering from egg allergy, preferably egg allergy. The egg allergy may be an allergy to egg white protein, or an allergy to mature egg protein (mature egg white protein or mature whole egg protein). The cooked egg protein may be baked.

The egg white protein formulations and dosage forms described herein are useful for treating human patients allergic to eggs, preferably chicken eggs, using oral immunotherapy. The egg allergy may be an allergy to egg white protein, or an allergy to mature egg protein (mature egg white protein or mature whole egg protein, such as baked egg white protein or baked whole egg protein). The most abundant allergenic proteins present in egg white are ovalbumin, ovomucoid, ovotransferrin and lysozyme, and patients may be allergic to one or more of these allergenic proteins.

Methods for diagnosing egg allergy are known in the art. For example, a patient may be diagnosed with a Skin Prick Test (SPT), an egg white protein specific IgE (ew-IgE) level, an oral food challenge, or a correlation of one or more allergic responses to egg protein consumption. For example, in some embodiments, the patient has about 0.35kU_Aa,/L or higher, about 0.7kU_Aa,/L, of about 3.5kU_Aa,/L or higher, about 5kU_Aa/L or higher, or about 7kU_ASerum ew-IgE levels of/L or higher. Serum ew-IgE levels can be determined using quantitative immunoassays. Quantitative immunoassays are known in the art and may include, but are not limited to, enzyme-linked immunosorbent assays (ELISAs); alkaline phosphatase immunoassay autoanalyzer, such asSystems (Siemens Healthcare Diagnostics, Erlangen, Germany); radioactive allergen adsorption test (RAST) or automatic fluorogenic enzyme immunoassay analyzer, such asSystem (Thermo Fisher Scientific/Phadia, Uppsala, Sweden). An automated fluorometric enzyme immunoassay analyzer is the preferred method.

Patients are typically about 4 years or older, such as between about 4 and 26 years of age.

Prior to initiation of treatment, by oral food challenge, a baseline highest tolerated dose or a baseline cumulative tolerated dose of raw egg white protein or cooked egg protein (cooked egg white protein or cooked whole egg protein, such as baked egg white protein or baked whole egg protein) of the patient can be determined. Oral food challenges involve 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 administered dose is separated 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), described in Sampson et al, Standard double blind, placebo controlled oral food challenges of American Academy of Allergy, Ashma & Immunology European Academy of Allergy and Clinical Immunology PRACTIC reports, J.Allergy Clin.Immunol., Vol.130, No. 6, pp.1260-74 (2012).

An oral food challenge directed against raw egg white protein to determine a highest tolerated dose or a 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, between about 1mg and about 2000mg, or between about 1mg to about 300 mg. Patients do not need to ingest all doses of oral food challenges, as oral food challenges typically terminate after patients suffer from moderate or severe allergenic adverse events. An exemplary set of raw egg white doses for oral food challenge is shown in table 1. For the exemplary oral food challenge doses shown in table 1, if the patient tolerates a maximum dose of egg white protein of 30mg (the highest tolerated dose), the cumulative tolerated dose is 44mg egg white protein.

Table 1: raw egg white protein dosage for oral food challenge

Raw ovalbumin dosage (mg)	Cumulative dose (mg)
		1	1
3	4
		10	14
30	44
		100	144
300	444
		600	1044
1000	2044
		2000	4044

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

The oral food challenge of cooked egg protein or baked egg protein may also be used to determine the highest tolerated dose of cooked egg protein or baked egg protein. The cooked or baked egg protein may be from cooked or baked egg white, or cooked or baked whole egg (i.e., egg white and egg yolk). For oral food challenges, a range of doses of cooked egg protein (which may be cooked, for example, in a baked food product such as bread, muffins, biscuits or cakes, or fried) is used. Table 2 shows an exemplary set of cooked or baked egg doses with doses of cooked whole egg protein in baked muffins for oral food challenge. In some embodiments, the baked egg protein or baked egg white protein is heated at about 176 ℃ to about 260 ℃ (such as about 176 ℃ to about 232 ℃, about 176 ℃ to about 220 ℃, or about 176 ℃ to about 205 ℃) for about 20 minutes to about 60 minutes (such as about 20 minutes to about 45 minutes, about 20 minutes to about 35 minutes, or about 20 minutes to about 30 minutes).

Table 2: cooked whole egg protein dosage for oral food challenge

Dosage numbering	Cooked/baked egg protein dosage (mg)	Cumulative dose (mg)
			1	125	125
2	250	375
			3	500	875
4	500	1375
			5	625	2000

While some patients treated with the oral immunotherapy described herein are able to tolerate mature egg proteins, some patients are unable to tolerate any or large amounts of mature or baked egg proteins. In some embodiments, the patient tolerates a maximum dose of mature or baked egg protein (i.e., the maximum tolerated dose) at the start of treatment of about 625mg or less, about 500mg or less, about 250mg or less, or about 125mg or less. In some embodiments, the highest cumulative dose of mature or baked egg protein (i.e., the highest tolerated dose) that the patient tolerates at the onset of treatment is about 2000mg or less, about 1375mg or less, about 875mg or less, about 375mg or less, or about 125mg or less.

Oral immunotherapy treatment schedule

Treating egg allergy in a patient by orally administering to the patient a plurality of doses of a pharmaceutical composition comprising an egg white protein according to an Oral Immunotherapy (OIT) schedule. OIT schedules typically include an up-dosing phase followed by a maintenance phase. Optionally, the OIT schedule further includes an initial increment phase that occurs before the start of the upregulation dose phase.

The doses may be provided in one or more dose containers (e.g., capsules or sachets) containing the pharmaceutical composition (also referred to herein as an "egg white protein preparation"). The dosage container is typically not ingested in its entirety, but is typically opened prior to administration and the pharmaceutical composition contained therein is consumed orally. The pharmaceutical composition may be mixed with a food vehicle to be consumed by the patient. Exemplary food vehicles include applesauce, pudding, oatmeal, beverages (e.g., milkshakes), or any other suitable food product mixed with a pharmaceutical composition.

Up regulating dosage stage

The up-dosing phase of the OIT schedule includes orally administering to the patient a series of increasing daily doses of ovalbumin. Egg white proteins are typically raw, but they may be pasteurized or otherwise processed in a manner that does not affect the presentation of protein epitopes. The egg white protein is contained within an egg white protein preparation, which can be prepared, for example, according to the methods described herein. The dose of egg white protein is preferably administered to the patient daily, but may be skipped, delayed, or delayed by a portion if the patient experiences one or more adverse events associated with administration of the egg white protein or a complication associated with increased sensitivity to an allergen not associated with administration of the egg white protein, such as atopic disease onset, inflammation, disease, menstruation, or unintended exposure of the patient to food to which the patient is allergic.

The serial ascending doses of egg white protein are generally in the range of about 1mg to about 300 mg. The given dose is administered daily (unless adjusted) to the patient for a period of at least two weeks before the dose is escalated to a higher dose. For example, due to an adverse event related to egg white protein or some other complication associated with increased sensitivity to an allergen not related to the egg white protein, escalating to higher doses may take longer than two weeks, which justifies delaying increasing the dose. The dose administered during the up-dosing phase is only incremented when the patient tolerates the previous dose. Thus, in some cases, the same dose may be administered to a patient for more than two weeks, such as three or more weeks, or four or more weeks. To ensure that the dose does not escalate too quickly, the up-dosing phase typically lasts for about 20 weeks or more, for example about 20 weeks to about 44 weeks.

The up-dosing phase may comprise administering to the subject 4, 5, 6, 7, 8, 9, or 10 or more different doses, which may range from about 1mg to about 300 mg. The different doses are spaced apart within the dosage range, but need not be evenly spaced apart. Exemplary doses that may be administered to a subject may include about 1mg, about 3mg, about 6mg, about 12mg, about 20mg, about 40mg, about 80mg, about 120mg, about 160mg, about 200mg, about 240mg, and about 300mg of egg white protein. In some embodiments, the up-dosing phase comprises administering a dose of at least 1mg and 300mg of egg white protein. In some embodiments, the maximum dose administered to the patient during the up-dosing phase is 300mg of egg white protein.

The escalated dose is preferably administered in a clinical setting (e.g., a physician's office, hospital, or other facility that allows immediate medical response upon the occurrence of a moderate or severe allergic adverse event). For example, if a patient has completed a daily administration of an 80mg dose of egg white protein for a period of two weeks, a 120mg dose of egg white protein may be administered in a clinical setting. If the patient tolerates the escalated dose, the following of that dose may be administered to the subject outside of the clinical setting, such as by the patient self-administering at home.

Maintenance phase

After the up-dosing phase, the OIT schedule includes a maintenance phase. The maintenance phase comprises administering to the patient a dose of ovalbumin for a period of time and is intended to maintain a desensitized state after the treatment is completed. Generally, the length of the maintenance phase is about 12 weeks or more, but it need not have a definite endpoint. In some embodiments, the maintenance dose is administered daily, although dosage administration may be adjusted, as discussed herein.

The dose of egg white protein administered during the maintenance phase (i.e., the "maintenance dose") is generally the highest tolerated dose that the patient obtained during the up-dosing phase, but in some cases the dose may be reduced. For example, if the patient successfully completes the up-dosing phase comprising administration of 300mg of egg white protein, the maintenance dose is typically 300mg of egg white protein, but may be reduced. For example, if the patient experiences one or more adverse events associated with administration of an egg white protein or a complication associated with increased sensitivity to an allergen not associated with administration of the egg white protein (such as atopic disease onset, inflammation, disease or menstruation), the maintenance dose may be reduced, skipped, delayed, or a portion delayed.

Initial incremental phase

Optionally, the initial escalation phase precedes the up-dosing phase in the OIT schedule. The initial escalation phase involves oral administration of a series of escalating doses of egg white protein to a patient over a day. The doses are separated by a period of time, typically about 20 minutes or more (such as about 20 minutes to about 60 minutes, or about 20 minutes to about 30 minutes). The time interval allows for monitoring the patient for adverse events associated with ovalbumin prior to administration of the next dose. Generally, the initial incremental phase occurs in a clinical setting.

The dose of egg white protein administered to a subject may be, for example, from about 0.2mg egg white protein to about 2mg egg white protein. The initial escalation phase may include 2, 3, 4, 5, 6 or more doses. Exemplary doses for administration to a subject may include about 0.2mg, about 0.4mg, about 0.8mg, about 1.2mg, and about 2mg of egg white protein. In another exemplary embodiment, the dose administered to the subject may include about 0.2mg, about 0.4mg, about 0.8mg, about 1.0mg, and about 2mg of egg white protein.

Dose adjustment

The oral immunotherapy schedule may be adjusted slightly if the patient experiences adverse events associated with administration of a dose of egg white protein (i.e., an allergic response to the dose) or complications associated with increased sensitivity to allergens not associated with administration of the dose of egg white protein. The adverse event associated with administration of the egg white protein may be a mild allergenic adverse event, a moderate allergenic adverse event, or a severe allergenic adverse event. Such dose adjustment may occur during an up-dosing phase of the OIT schedule or a maintenance phase of the OIT schedule. The dose adjustment may be to reduce the dose, skip scheduled dose administration, delay part of the dose, or delay dose escalation. By adjusting the dosage regimen, the risk of experiencing a more severe adverse event (if the dosage is adjusted in response to an adverse event associated with administration of egg white protein) or experiencing an adverse event or severity of an adverse event (if the dosage is adjusted in response to an adverse event not associated with administration of egg white protein) is reduced.

Adverse events associated with administration of egg white protein that may lead to dose adjustments may be, for example, hypersensitivity, allergy (e.g., anaphylactic shock), or gastrointestinal symptoms (such as abdominal pain or vomiting). Figure 4 illustrates an exemplary decision tree for adjusting dosage based on severity of adverse events associated with administration of ovalbumin.

A complication associated with increased sensitivity to an allergen that may lead to dose adjustments may be any event or patient state that occurs concurrently with administration of the pharmaceutical composition that will enhance the sensitivity of the patient to the allergen. This may be due to, for example, immunological changes caused by concurrent factors associated with increased sensitivity to allergens. Exemplary complications associated with increased sensitivity to allergens include the onset of atopic diseases (e.g., eczema, asthma, or rhinoconjunctivitis), allergic reactions to allergens other than ovalbumin, inflammation (e.g., due to surgery or traumatic injury), diseases (such as infection), and menstruation. A complication may also be an unintended exposure (e.g., consumption) of the patient to food to which the patient is allergic. The food to which the patient is allergic may be eggs, or may be some other food to which the patient is also allergic, such as milk, peanuts, tree nuts, wheat, shellfish or soy.

The dosage of the pharmaceutical composition can be adjusted by reducing the dosage of the pharmaceutical composition. Dose reduction is temporary and may increase over a period of time, for example after an adverse event or complication associated with increased sensitivity to an allergen has elapsed. The dose may be reduced by up to about 50%, or to less than 50%, of the next available dose relative to the previously administered dose. In some embodiments, the dose is reduced by 1 dose level or 2 dose levels from a previously administered dose. In some embodiments, one or more (such as a first) reduced doses are administered in a clinical setting. Once the dose is reduced, the reduced dose can be administered to the patient for about one week or more (such as two weeks or more, between about one week and about four weeks, or between about one week and about two weeks) before incrementing subsequent doses. The ascending dose is preferably administered in a clinical setting such that adverse events associated with administration of the pharmaceutical composition to a patient can be monitored. Administration of increasing doses should be attempted, but if an allergic response results, administration of decreasing doses to the patient may continue.

In some embodiments, the dose is adjusted by skipping administration of one or more predetermined doses. For example, if daily administration of the dose is planned, one, two, three or more daily doses may be skipped before administration of the pharmaceutical composition is resumed. Administration of the pharmaceutical composition may be the same dose as previously administered or resumed at a reduced dose. If more than one dose is skipped, it is generally preferred that the next dose is administered to the patient in a clinical setting.

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

Dose administration may also be adjusted by delaying a predetermined increment of the dose administered to the patient during the up-dosing phase. The OIT schedule typically has a preset increment time, such as once every two weeks. However, if the patient is experiencing an adverse event or a complication associated with increased sensitivity to an allergen, increasing the dose level may increase the risk of an adverse event or a more severe adverse event. Thus, incrementing may be delayed, for example, until the adverse event or complication subsides. In some embodiments, the increment may be delayed by about one week or more, about two weeks or more, or about three weeks or more.

In one example, a method of adjusting a dose of a pharmaceutical composition comprising egg white protein during oral immunotherapy for egg allergy in a subject, the method comprising orally administering a first dose of the pharmaceutical composition to a patient; and orally administering a second dose of the pharmaceutical composition to the patient, wherein the second dose is reduced, skipped, or at least a portion of the dose is delayed if the patient experiences an adverse event associated with administration of the first dose; wherein the oral immunotherapy comprises (i) an up-dosing phase comprising orally administering to the patient a series of ascending doses of egg white protein, and (ii) a maintenance phase comprising orally administering to the patient a plurality of maintenance doses comprising egg white protein; the method comprises the following steps: delaying the second dose can include dividing the second dose into a first portion and a second portion if the patient experiences an adverse event related to administration of the first dose, wherein the first portion is administered according to a predetermined dosing schedule, and wherein the second portion is delayed relative to the predetermined dosing schedule (e.g., by about 8 hours to about 16 hours, or by about 8 hours to about 12 hours). 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 an up-dosing phase, and in some embodiments, the first dose and the second dose are administered to the patient during a maintenance phase.

In another example, a method of adjusting a dose of a pharmaceutical composition comprising egg white protein during oral immunotherapy for egg allergy in a subject, the method comprising orally administering a first dose of the pharmaceutical composition to a patient; and orally administering a second dose of the pharmaceutical composition to the patient, wherein the second dose is reduced or skipped if the patient experiences a complication associated with increased sensitivity to a stressor that is not associated with administration of the first dose, wherein the oral immunotherapy comprises (i) an up-dosing phase comprising orally administering a series of ascending doses of ovalbumin protein to the patient, and (ii) a maintenance phase comprising orally administering a plurality of maintenance doses comprising ovalbumin protein to the patient; the method comprises the following steps: complications associated with increased sensitivity to allergens may be, for example, atopic disease episodes, inflammation, disease, or menstruation. In some embodiments, the first dose and the second dose are administered to the patient during an up-dosing phase of the oral immunotherapy, and in some embodiments, the first dose and the second dose are administered to the patient during a maintenance phase of the oral immunotherapy.

Treatment endpoints for oral immunotherapy

The oral immunotherapy described herein is intended to desensitize patients to egg allergens to reduce the risk of adverse allergic events due to accidental exposure to egg white proteins. The degree of desensitization to the raw egg white protein and/or the 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 food challenges, as described above with reference to baseline characteristics of the patient. Successful treatment can be indicated by an increase in the highest tolerated dose of raw egg white protein or cooked (or baked) egg protein or an increase in the cumulative tolerated dose after treatment compared to when treatment was initiated.

In some embodiments, the patient is able to tolerate a dose of about 300mg of raw egg white protein, about 600mg of raw egg white protein, about 1000mg of raw egg white protein, or about 2000mg of raw egg white protein after the up-dosing phase is complete. In some embodiments, the patient is able to tolerate a dose of about 300mg of raw egg white protein, about 600mg of raw egg white protein, about 1000mg of raw egg white protein, or about 2000mg of raw egg white protein after the maintenance phase is over. In some embodiments, the patient is able to tolerate a cumulative dose of about 444mg raw egg white protein, about 1044mg raw egg white protein, about 2044mg raw egg white protein, or about 4044mg raw egg white protein at the end of the up-dosing phase. In some embodiments, the patient is able to tolerate a cumulative dose of about 444mg raw egg white protein, about 1044mg raw egg white protein, about 2044mg raw egg white protein, or about 4044mg raw egg white protein at the end of the maintenance phase.

In some embodiments, when the patient cannot tolerate a dose of about 625mg cooked (or baked) egg protein, about 500mg cooked (or baked) egg protein, about 250mg cooked (or baked) egg protein, or about 125mg cooked (or baked) egg protein at the beginning of the treatment, the patient can tolerate a dose of about 625mg cooked (or baked) egg protein at the end of the up-dosing phase. In some embodiments, the patient is able to tolerate a dose of about 625mg cooked (or baked) egg protein at the end of the maintenance phase when the patient is unable to tolerate a dose of about 625mg cooked (or baked) egg protein, about 500mg cooked (or baked) egg protein, about 250mg cooked (or baked) egg protein, or about 125mg cooked (or baked) egg protein at the start of treatment. In some embodiments, the patient is able to tolerate a cumulative dose of about 2000mg cooked (or baked) egg protein at the end of the up-dosing phase, wherein the patient is unable to tolerate a cumulative dose of about 2000mg cooked (or baked) egg protein, about 1375mg cooked (or baked) egg protein, about 875mg cooked (or baked) egg protein, about 375mg cooked (or baked) egg protein, or about 125mg cooked (or baked) egg protein at the beginning of the treatment. In some embodiments, the patient is able to tolerate a cumulative dose of about 2000mg cooked (or baked) egg protein at the end of the maintenance phase, wherein the patient is unable to tolerate a cumulative dose of about 2000mg cooked (or baked) egg protein, about 1375mg cooked (or baked) egg protein, about 875mg cooked (or baked) egg protein, about 375mg cooked (or baked) egg protein, or about 125mg cooked (or baked) egg protein at the beginning of the treatment.

Exemplary embodiments

The following embodiments are exemplary and should not be considered as limiting the invention described herein.

Embodiment 1: a method of preparing an egg white protein formulation, the method comprising:

(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 at a higher shear force than the shear force used for mixing in step (c); and

(f) mixing the third mixture with a lubricant to form the egg white protein formulation.

Embodiment 2: the method of embodiment 1, wherein the egg white protein formulation has about 0.05% to about 2.5% egg white protein by weight.

Embodiment 3: the method of embodiment 1 or 2, wherein the egg white protein formulation has about 0.1% to about 0.7% egg white protein by weight.

Embodiment 4: the method of any of embodiments 1-3, wherein step (b) comprises passing at least a portion of the second amount of the first diluent and the first mixture through the mesh screen.

Embodiment 5: the method of any of embodiments 1-3, wherein step (b) comprises passing the second amount of the first diluent and the first mixture through the mesh screen.

Embodiment 6: the method according to any one of embodiments 1-5, wherein step (c) comprises a plurality of sub-steps, wherein each sub-step comprises (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 with the first mixture.

Embodiment 7: the method of embodiment 6, wherein step (c) comprises three or more substeps.

Embodiment 8: the method of any of embodiments 1-5, wherein step (c) comprises continuously mixing the first mixture and the second amount of the first diluent while the second amount of the first diluent is added to the first mixture.

Embodiment 9: the method of any of embodiments 1-8, wherein the first mixture and the second amount of the first diluent are mixed in a drum blender.

Embodiment 10: the method of any of embodiments 1-9, wherein the second mixture is mixed with the second diluent in a drum blender.

Embodiment 11: the method of any of embodiments 1-10, wherein the third mixture is mixed using a cone mill.

Embodiment 12: the method of any of embodiments 1-11, wherein the third mixture is mixed with the lubricant in a drum blender.

Embodiment 13: the method of any of embodiments 1-12, wherein a third amount of the first diluent is mixed with the third mixture.

Embodiment 14: the method of embodiment 13, wherein the third amount of the first diluent and the lubricant are co-mixed with the third mixture.

Embodiment 15: the method of any of embodiments 1-14, comprising mixing the lubricant with an additional portion of the first diluent or the second diluent prior to mixing the lubricant with the third mixture.

Embodiment 16: the method of embodiment 15, wherein the mixture of the lubricant and additional portions of the first diluent or the second diluent is passed through a mesh screen prior to mixing the lubricant with the third mixture.

Embodiment 17: a method of preparing an egg white protein formulation, the method comprising:

(a) mixing a dry egg white protein powder with a first amount of a first diluent to form a first mixture;

(b) co-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; and

(f) mixing the fourth mixture with the fifth mixture.

Embodiment 18: the method of embodiment 17, comprising mixing the second mixture prior to step (c).

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

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

Embodiment 21: the method of any one of embodiments 1-20, wherein the egg white protein preparation is substantially free of colloidal silica.

Embodiment 22: the method of any one of embodiments 1-21, wherein the egg white protein formulation has from about 40% to about 70% by weight of the first diluent.

Embodiment 23: the method of any one of embodiments 1-22, wherein the egg white protein formulation has from about 30% to about 50% by weight of the second diluent.

Embodiment 24: the method of any one of embodiments 1-23, wherein the egg white protein formulation has from about 0.1% to about 2% by weight of the lubricant.

Embodiment 25: a method of preparing an egg white protein formulation, the method comprising:

(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;

(d) mixing the third mixture at a higher shear force than the shear force used for mixing in step (b); and

(e) Mixing the third mixture with a lubricant to form the egg white protein formulation.

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

Embodiment 27: the method of embodiment 25 or 26, wherein the egg white protein formulation has about 1% to about 70% egg white protein by weight.

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 cone mill.

Embodiment 29: the method according to any one of embodiments 25-28, wherein step (b) comprises two mixing sub-steps, wherein one mixing sub-step is at a higher shear force than the other mixing sub-step.

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 the third mixture.

Embodiment 31: the method of any one of embodiments 25-29, wherein step (c) further comprises co-mixing the second mixture with additional amounts of the first diluent and the second diluent to form the third mixture.

Embodiment 32: the method of any one of embodiments 25-31, mixing the third mixture in step (d) using a cone mill.

Embodiment 33: the method of any of embodiments 25-32, wherein the second amount of the first diluent and the first mixture are mixed in a drum blender.

Embodiment 34: the method of any of embodiments 25-33, wherein the second mixture is mixed with the second diluent in a drum blender.

Embodiment 35: the method of any of embodiments 25-33, wherein a third amount of the first diluent is mixed with the third mixture.

Embodiment 36: the method of embodiment 35, wherein the third amount of the first diluent and the lubricant are co-mixed with the third mixture.

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 (i) said portion of said mixture 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: the method of any of embodiments 25-37, wherein the third mixture is mixed with the lubricant in a drum blender.

Embodiment 39: the method of any one of embodiments 25-38, wherein the egg white protein formulation has about 9% to about 85% by weight of the first diluent.

Embodiment 40: the method of any one of embodiments 25-39, wherein the egg white protein formulation has from about 10% to about 50% by weight of the second diluent.

Embodiment 41: the method of any one of embodiments 25-40, wherein the egg white protein formulation has from about 10% to about 20% by weight of the second diluent.

Embodiment 42: the method of any one of embodiments 25-41, wherein the egg white protein formulation has from about 0.1% to about 2% by weight of the lubricant.

Embodiment 43: the method of any one of embodiments 25-42, wherein the egg white protein formulation comprises a glidant.

Embodiment 44: the method of embodiment 43, wherein the glidant 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 glidant is colloidal silicon dioxide.

Embodiment 46: the method of any one of embodiments 25-45, wherein the egg white protein formulation has about 2% to about 70% egg white protein by weight.

Embodiment 47: the method of any one of embodiments 25-44, wherein the egg white protein preparation is substantially free of colloidal silica.

Embodiment 48: the method of embodiment 47, wherein the egg white protein formulation has about 1% to about 5% egg white protein by weight.

Embodiment 49: a method of preparing an egg white protein formulation, the method comprising:

(a) mixing a dry egg white protein powder, a first diluent, and a glidant to form a first mixture;

(b) mixing a second diluent with the first mixture at a higher shear force than the shear force used for mixing in step (a) to form a second mixture;

Embodiment 50: the method of embodiment 49, wherein the egg white protein formulation has about 50% to about 80% egg white protein by weight.

Embodiment 51: the method of embodiment 49 or 50, wherein the dried egg white protein powder, the first diluent, and the glidant are mixed in a drum blender.

Embodiment 52: the method of any of embodiments 49-51, wherein the second diluent and the first mixture are mixed in step (b) using a cone mill.

Embodiment 53: the method of any of embodiments 49-52, wherein the second mixture and the lubricant are mixed in a drum blender.

Embodiment 54: the method of any of embodiments 49-53, wherein a second amount of the first diluent is mixed with the second mixture.

Embodiment 55: the method of embodiment 54, wherein the second amount of the first diluent and the lubricant are co-mixed with the second mixture.

Embodiment 56: the method of any of embodiments 49-55, wherein the glidant comprises colloidal silicon dioxide.

Embodiment 57: the method according to 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: the method of any one of embodiments 1-57, wherein the egg white protein formulation is prepared in a batch size of about 5kg or greater.

Embodiment 59: the method of embodiment 58, wherein said egg white protein preparation is prepared in a batch size of about 5kg to about 50 kg.

Embodiment 60: the method of any one of embodiments 1-59, comprising determining an egg white protein blend uniformity of the egg white protein formulation.

Embodiment 61: the method of any one of embodiments 1-60, wherein the egg white protein formulation has an egg white protein blend uniformity Relative Standard Deviation (RSD) of about 15% or less.

Embodiment 62: the method of any one of embodiments 1-61, comprising packaging the egg white protein formulation in a plurality of dosage containers.

Embodiment 63: the method of embodiment 62, wherein the dose container is a capsule or a pouch.

Embodiment 64: the method of embodiment 62 or 63, comprising determining ovalbumin content uniformity for the plurality of dosage containers.

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

Embodiment 66: the method of any one of embodiments 1-65, wherein the first diluent is pregelatinized starch.

Embodiment 67: the method of any of embodiments 1-66, wherein the second diluent is microcrystalline cellulose.

Embodiment 68: the method of any one of embodiments 1-67, wherein the lubricant is magnesium stearate.

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

Embodiment 70: the method of any one of embodiments 1-69, wherein the forming of the dried egg white protein powder comprises spray drying liquid egg white.

Embodiment 71: the method of any one of embodiments 1-70, wherein the egg white protein powder has been depleted of glucose.

Embodiment 72: the method according to any one of embodiments 1-71, wherein said dried egg white protein powder has been pasteurized.

Embodiment 73: the method of any one of embodiments 1-72, wherein the dried egg white protein powder is derived from an egg.

Embodiment 74: the method according to any one of embodiments 1-73, further comprising characterizing ovomucoid, ovalbumin, ovotransferrin, or lysozyme in the dried egg white protein powder.

Embodiment 75: the method according to any one of embodiments 1-74, further comprising characterizing ovomucoid, ovalbumin, ovotransferrin, or lysozyme in the egg white protein formulation.

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.

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

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

Embodiment 79: the method according to any one of embodiments 76-78, comprising comparing the obtained HPLC profile with a reference HPLC profile.

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

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 compared to the total protein in the egg white protein powder or the 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 compared to the total amount of ovomucoid, ovalbumin, ovotransferrin, or lysozyme in the egg white protein powder or the egg white protein formulation.

Embodiment 83: the method of any one of embodiments 74-82, wherein characterizing ovomucoid, ovalbumin, ovotransferrin, or lysozyme comprises measuring the potency of the ovomucoid, ovalbumin, ovotransferrin, or lysozyme in the egg white protein powder or the 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 the egg white protein formulation 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 the potency of ovomucoid, ovalbumin, ovotransferrin, or lysozyme is measured using an immunoassay.

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

Embodiment 87: the method of embodiment 85, wherein said immunoassay comprises the use of an antibody library 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.

Embodiment 88: the method of embodiments 86 and 87, wherein the antibody is an IgE antibody or an IgG antibody.

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

Embodiment 90: an egg white protein formulation prepared according to the method of any one of embodiments 1-89.

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.

Embodiment 92: the egg white protein formulation of embodiment 91, wherein the egg white protein formulation comprises about 0.1% to about 3.5% by weight egg white protein.

Embodiment 93: an egg white protein formulation according to embodiment 91 or 92 wherein the first diluent is pregelatinized starch.

Embodiment 94: an egg white protein formulation according to any of embodiments 91-93 wherein the second diluent is microcrystalline cellulose.

Embodiment 95: the egg white protein formulation of any one of embodiments 91-94, wherein the lubricant is magnesium stearate.

Embodiment 96: the egg white protein formulation of any one of embodiments 91-95, wherein the egg white protein formulation consists essentially of the egg white protein powder, the first diluent, the second diluent, and the lubricant.

Embodiment 97: a method of treating egg allergy in a patient, comprising:

orally administering to the patient a plurality of doses of a pharmaceutical composition comprising an egg white protein according to an oral immunotherapy schedule comprising:

Embodiment 98: the method of embodiment 97, wherein the patient has an egg white-specific serum IgE (ew-IgE) level of about 7kUA/L or higher at the start of treatment.

Embodiment 99: the method of embodiment 97, wherein the patient has an egg white-specific serum IgE (ew-IgE) level of about 5kUA/L or higher at the start of treatment.

Embodiment 100: the method of any one of embodiments 97-99, wherein the series of escalating doses administered to the patient during the up-dosing phase comprises at least a 1mg dose of egg white protein and a 300mg dose of egg white protein.

Embodiment 101: the method of any one of embodiments 97-100, wherein the series of escalating doses administered to the patient during the upregulating dose phase comprises at least 10 different doses of egg white protein.

Embodiment 102: the method of any one of embodiments 97-101, wherein the series of escalating doses administered to the patient during the up-dosing phase comprises doses of about 1mg, about 3mg, about 6mg, about 12mg, about 20mg, about 40mg, about 80mg, about 120mg, about 160mg, about 200mg, about 240mg, and about 300mg ovalbumin.

Embodiment 103: the method according to any one of embodiments 97-102, wherein the dose administered during the upregulating dose phase is only incremented when the patient tolerates the previous dose.

Embodiment 104: the method of any one of embodiments 97-103, wherein the maximum dose administered to the patient during the upregulating dosage phase is about 300mg egg white protein.

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

Embodiment 106: the method of any one of embodiments 97-105, wherein the maintenance dose administered to the patient during the maintenance phase is about 300mg egg white protein.

Embodiment 107: the method according to any one of embodiments 97-106, wherein the maintenance dose is administered to the patient only if the patient tolerates the maximum dose administered to the patient during the upregulating dose phase.

Embodiment 108: the method according to any one of embodiments 97-107, wherein the patient tolerates a dose of about 600mg of raw egg white protein at the end of the maintenance phase.

Embodiment 109: the method according to any one of embodiments 97-108, wherein the patient tolerates a dose of about 1000mg of raw egg white protein at the end of the maintenance phase.

Embodiment 110: the method according to any one of embodiments 97-109, wherein the patient tolerates a dose of about 2000mg of raw egg white protein at the end of the maintenance phase.

Embodiment 111: the method of any one of embodiments 97-110, wherein the patient tolerates a cumulative dose of about 2000mg mature egg white protein at the end of the maintenance phase.

Embodiment 112: the method according to any one of embodiments 97-111, wherein the patient tolerates a cumulative dose of about 2000mg baked ovalbumin protein at the end of the maintenance phase.

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

Embodiment 114: the method according to any one of embodiments 97-113, wherein the patient is unable to tolerate a cumulative dose of about 2000mg of mature egg white protein prior to initiation of treatment.

Embodiment 115: the method according to any one of embodiments 97-114, wherein the patient is unable to tolerate a cumulative dose of about 2000mg of baked ovalbumin protein prior to initiation of treatment.

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

Embodiment 117: the method according to any one of embodiments 97-113 and 116, wherein the patient tolerates a cumulative dose of about 2000mg baked ovalbumin protein prior to initiation of treatment.

Embodiment 118: the method of any one of embodiments 97-117, wherein the oral immunotherapy schedule comprises an initial escalation phase prior to the escalation dosage phase, the initial escalation phase comprising orally administering to the patient a series of escalating doses of about 0.2mg to about 2mg of egg white protein over a day, wherein the patient is administered a single administration of any given dose, and wherein the doses are separated by at least 15 minutes.

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

Embodiment 120: the method of any one of embodiments 97-119, wherein the patient is about 4 years or older before the treatment begins.

Embodiment 121: the method of any one of embodiments 97-120, wherein the patient is about 4 to about 26 years old before treatment begins.

Embodiment 122: a method of adjusting a dose of a pharmaceutical composition comprising an egg white protein during oral immunotherapy for egg allergy in a subject, the oral immunotherapy comprising (i) an up-dosing phase comprising orally administering a series of ascending doses of the egg white protein to the patient, and (ii) a maintenance phase comprising orally administering a plurality of maintenance doses comprising the egg white protein to the patient; the method comprises the following steps:

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 the second dose is reduced, skipped, or at least part of the dose is delayed if the patient experiences an adverse event related to administration of the first dose.

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

Embodiment 124: the method of embodiment 123, wherein the second part is delayed from about 8 hours to about 12 hours after administration of the first part.

Embodiment 125: the method of embodiment 122, wherein the second dose is skipped if the patient experiences an adverse event related to administration of the first dose.

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 related to administration of the first dose.

Embodiment 127: the method of embodiment 126, wherein prior to incrementing a subsequent dose of the pharmaceutical composition, the subsequent dose is reduced relative to the first dose by about one week or more.

Embodiment 128: the method of embodiment 126 or 127, wherein prior to attempting to increment a subsequent dose of the pharmaceutical composition, the subsequent dose is reduced relative to the first dose by about one week to about two weeks.

Embodiment 129: the method of any one of embodiments 126-128, wherein prior to incrementing a subsequent dose of the pharmaceutical composition, the subsequent dose is reduced relative to the first dose by about one week to about two weeks.

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

Embodiment 131: the method of any one of embodiments 122-130, wherein the adverse event associated with the administration of the first dose is a moderate allergenic adverse event or a severe allergenic adverse event.

Embodiment 132: the method according to any one of embodiments 122-131, wherein the first dose and the second dose are administered to the patient during the up-dosing phase of the oral immunotherapy.

Embodiment 133: the method according to any one of embodiments 122-131, wherein the first dose and the second dose are administered to the patient during the maintenance phase of the oral immunotherapy.

Embodiment 134: a method of adjusting a dose of a pharmaceutical composition comprising an egg white protein during oral immunotherapy for egg allergy in a subject, the oral immunotherapy comprising (i) an up-dosing phase comprising orally administering a series of ascending doses of the egg white protein to the patient, and (ii) a maintenance phase comprising orally administering a plurality of maintenance doses comprising the egg white protein to the patient; the method comprises the following steps:

orally administering a first dose of the pharmaceutical composition to the patient; and

Embodiment 135: the method of embodiment 134, wherein the complication associated with increased sensitivity to an allergen is atopic seizure, inflammation, illness, or menstruation.

Embodiment 136: the method of embodiment 134 or 135, wherein the second dose is skipped if the patient experiences a complication associated with increased sensitivity to an allergen not associated with administration of the first dose.

Embodiment 137: the method of embodiment 134 or 135, wherein the second dose is reduced relative to the first dose if the patient experiences a complication associated with increased sensitivity to an allergen not associated with administration of the first dose.

Embodiment 138: the method of embodiment 137, wherein prior to incrementing a subsequent dose of the pharmaceutical composition, the subsequent dose is reduced relative to the first dose by about one week or more.

Embodiment 139: the method of embodiment 137 or 138, wherein prior to attempting to increment a subsequent dose of the pharmaceutical composition, the subsequent dose is reduced relative to the first dose by about one week to about two weeks.

Embodiment 140: the method of any one of embodiments 137-139, wherein prior to incrementing a subsequent dose of the pharmaceutical composition, the subsequent dose is reduced relative to the first dose by about one week to about two weeks.

Embodiment 141: the method according to any one of embodiments 134-140, wherein the first dose and the second dose are administered to the patient during the up-dosing phase of the oral immunotherapy.

Embodiment 142: the method according to any one of embodiments 134-141, wherein the first dose and the second dose are administered to the patient during the maintenance phase of the oral immunotherapy.

Embodiment 143: the method according to any one of embodiments 134-142, wherein the complication associated with increased sensitivity to an allergen is an unintended exposure of the patient to a food to which the patient is allergic.

Embodiment 144: the method of any one of embodiments 97-143, wherein the egg white protein in the pharmaceutical composition is a raw egg white protein.

Embodiment 145: the method of any one of embodiments 97-144, wherein the pharmaceutical composition is mixed with a food vehicle prior to administration.

Embodiment 146: a pharmaceutical composition for use in a method according to any one of embodiments 97-145.

Embodiment 147: the pharmaceutical composition of embodiment 146, wherein the pharmaceutical composition comprises an egg white protein formulation prepared according to the method of any one of embodiments 1-96.

Embodiment 148: a pharmaceutical composition for use in the preparation of a medicament for use in a method of treating egg allergy in a patient according to the method of any one of embodiments 97-145.

Embodiment 149: the pharmaceutical composition of embodiment 148, wherein the pharmaceutical composition comprises an egg white protein formulation prepared according to the method of any one of embodiments 1-96.

Examples

The present application may be better understood by reference to the following non-limiting examples provided as exemplary embodiments of the present application. The following examples are given to more fully illustrate the embodiments, however, should in no way be construed as limiting the broad scope of the application. While certain embodiments of the present application have been shown and described herein, it will be obvious that such embodiments are provided by way of example only. Numerous modifications, changes, and substitutions will 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 employed in practicing the methods described herein.

Example 1: characterization of dried ovalbumin powder

Many dried egg white protein powders were obtained from commercial suppliers and analyzed to assess the quality of the powder used to prepare the egg white protein formulations. Certain characterization methods include comparing samples from one or more batches of dried egg white protein powder to a reference standard. The reference standard was a sample of dried egg white protein powder monitored over time and found to have stable characteristics.

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

Table 3: total protein in a drug substance batch

	Batch A	Batch B
			Protein content (% by weight)	75％	75％

Size exclusion chromatography (SEC-HPLC) was used as one method to establish HPLC profiles of allergenic proteins, as well as an orthogonal method to determine the protein content of dry egg white protein powder. The mobile phase was phosphate buffered saline solution (ph6.8), which was also used to dissolve the dried egg white powder samples. The absorbance of the column eluate was measured at 220 nm. The identity of the peaks has been previously determined by injection of four commercially available purified reference products of egg white allergenic proteins: ovalbumin (OVA), Ovomucoid (OVM), Ovotransferrin (OVT), and Lysozyme (LYS). The SEC-HPLC chromatogram is shown in fig. 5, where ovotransferrin, ovomucoid, ovalbumin and lysozyme peaks were identified. The remaining peaks have not been identified. Table 4 gives the peak area percentages of the four egg white allergen peaks of the two batches of dried egg white protein powder. Although the ovomucoid and ovalbumin proteins have different molecular weights determined from their amino acid sequences, they are co-eluted using this SEC-HPLC method. This is probably due to protein glycosylation and protein structure giving them similar apparent molecular weights and retention times.

Table 4: percentage of peak area in SEC-HPLC chromatogram

Egg white protein	Batch C	Batch D
			Ovotransferrin	11	13
Ovalbumin and ovomucoid	66	75
			Lysozyme	3.4	Not integrated

In order to improve resolution and allow physical separation of allergens in egg white, reverse phase HPLC (RP-HPLC) methods were developed. RP-HPLC method based on the use of wide poresReversed phase separation of 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 the allergenic protein in the dried ovalbumin powder. Data were collected with a UV detector at a wavelength of 210 nm. Chromatographic peaks were identified by comparing chromatographic peaks from dried egg white protein powder samples with commercially purified ovotransferrin, ovalbumin, ovomucoid, and lysozyme protein standards. As shown in FIG. 6, the RP-HPLC resolved the ovotransferrin, ovalbumin, ovomucoid, and lysozyme chromatogram peaks. The relative amounts of each of ovomucoid, lysozyme, ovotransferrin and ovalbumin (compared to the amount of total protein) in the dried egg white protein powder reference standard as well as the dried egg white protein powders of batch a and batch B as determined by chromatographic peak area are shown in table 5.

Table 5: relative amount of allergenic egg white protein (peak area percentage)

The allergenic egg white proteins were further characterized by SDS-PAGE. The proteins in the dried egg white protein powder were extracted with water, reduced with dithiothreitol, and separated using triglycine gel (4% -20% gradient). Protein bands were detected using coomassie blue staining. Purified egg white protein allergens (ovomucoid, ovalbumin, lysozyme and ovotransferrin) were obtained from commercial sources and used as comparative standards for analysis. The SDS-PAGE data (FIG. 7) confirmed the presence of 4 ovalbumin allergen components in the analyzed samples, providing additional protein profile data for SEC-HPLC and RP-HPLC data. In addition, the staining pattern and intensity of all bands was consistent from batch to batch. The loading of the SDS-PAGE gels shown in FIG. 7 is listed in Table 6.

Table 6: loading onto SDS-PAGE gel of FIG. 7 and immunoblotting of FIG. 8

Lane lane	1	2	3	4	5	6
							Sample (I)	Buffering agent	MWM	Batch A	Reference standard	Batch D	Buffering agent
Lane lane	7	8	9	10	11	12
							Sample (I)	OVM	OVA	LYS	OVT	MWM	Buffering agent

MWM, molecular weight marker; ref.std., reference standard; OVM, ovomucoid; OVA, ovalbumin; LYS, lysozyme; OVT, ovotransferrin.

Immunoblot analysis of the dried egg white protein powder was also performed to further characterize the allergenic egg white protein in the dried egg white protein powder. As SDS-PAGE, a gel was formed as described above (see Table 6). The 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 is then treated with pooled serum from rabbits immunized with one of ovalbumin, ovomucoid, ovotransferrin, or lysozyme. As shown in fig. 8, for the three dry egg white protein powders, the protein band distribution and intensity was consistent from batch to batch, with bands at the expected molecular weights and similar immunoreactivity patterns for the egg white protein allergens.

ELISA was used to determine the relative potency of the ovomucoid in the dried egg white protein powders from lot a and lot B relative to a reference standard. The proteins in the dried egg white protein powder samples were extracted in Phosphate Buffered Saline (PBS) at room temperature, vortexed, centrifuged, and filtered, then serially diluted and adsorbed onto ELISA plates. The plates were then blocked with 5% skim milk, washed, and incubated with primary antibody (rabbit anti-ovomucin IgG). In turn, the plate was washed again, a secondary antibody conjugated to horseradish peroxidase was introduced, and then TMB substrate was added. The extent of binding of anti-ovomucoid to ovomucoid in the sample is determined by a colorimetric reaction. The data is fitted with a logistic curve fitting formula (e.g., 4-parameter curve fitting), and the EC is determined ₅₀The value is obtained. EC (EC)₅₀The values reflect the potency of the ovomucoid in the dry egg white protein powder batch and are based on EC₅₀Ratio of values the relative potency of the dried egg white protein powder was calculated (table 7). Specifically, the 4-parameter curve fitting formula used is y ═ a-D)/(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 represents the half-maximal Effective Concentration (EC) corresponding to the intermediate response between the lower and upper asymptotes₅₀) And D is the maximum asymptote. By reference to EC of standard₅₀Divided by the EC of the sample₅₀And obtaining the relative efficiency of the sample. The reference standard may be, for example, a dried egg white protein powder from a previously validated batch or a previously validated batch of a pharmaceutical composition.

Table 7: relative potency of dried egg white protein powder batches

Batches of	Relative efficiency
		A	1.0
B	1.2

The particle size distribution of the dried egg white protein powder batch was measured using laser diffraction. Dry egg white protein powder was dispersed in methanol, pumped through a flow cell, and analyzed by laser diffraction. Measurement of D₅₀(median particle diameter by volume of particles), D₁₀(particle size at 10 th percentile, by particle volume) and D₉₀(particle size at the 90 th percentile, based on particle volume) and is given in table 8.

Table 8: particle size distribution of dried egg white protein powder

Particle size	Reference standard	Batch A	Batch B
				D₁₀(μm)	15	14	12
D₅₀(μm)	77	74	66
				D₉₀(μm)	170	166	162

The water activity of the dried egg white protein powder was measured by placing 5g of the dried egg white protein powder in a sample cup and placing in a water activity meter (AquaLab 4 TEV). In a sealed chamber of the instrument, the liquid and gas phases of water in the sample are equilibrated at 25 ℃ to obtain a_wI.e. the relative humidity of the headspace. The results of this measurement are shown in table 9.

Table 9: water activity of dried egg white protein powder

Example 2: evaluation of excipients for egg white protein formulations

During formulation and process development, five batches of egg white protein formulations were prepared and encapsulated in capsule dosage forms. The formulations were prepared to produce 200mg or 300mg doses in size 0 or 00 capsules. The contents and measured characteristics of each formulation are shown in table 10. The deliverable mass percentage of the egg white protein formulation was from the capsule sampling.

Table 10: summary of ovalbumin protein formulation and capsule characterization

When the capsules were reopened to deliver the dry egg white protein formulation contents for content uniformity testing, an average of about 6 to 9 wt% of the formulation remained adhered to the interior of the reopened capsules when no colloidal silica was included in the formulation (batches 1-3). In addition, online observations were recorded during the encapsulation of these batches, i.e., the dry egg white protein formulation remained adhered within both ends of the re-opened capsule shell when the capsule was re-opened and shaken to simulate delivery of the powder contents.

Colloidal silicon dioxide was subsequently added as a glidant to a 300mg dose strength blend formulation to improve powder flow and deliverable mass from the capsule shell during administration. The addition of colloidal silica results in a significant improvement in the ease with which the powder can be delivered from the shell of a pull-apart capsule. For subsequent formulation and process development lots (lots 4-6) at 300mg dose strength, the average deliverable mass ranged from 99 wt% to 100 wt%. The delivered powder from the reopened capsule was free flowing with some soft agglomerates (due to packing during encapsulation) that turned into powder when lightly pinched between two fingers.

Example 3: preparation of egg white protein preparation

Dried egg white protein preparations were prepared for dosage forms having the label claims of 0.2mg, 1mg, 6mg, 12mg and 300mg, and dried egg white protein preparations were prepared in 7kg batches for dosage forms having the label claims of 3mg, 20mg, 40mg, 80mg, 120mg, 160mg, 200mg and 240 mg. The nominal protein content of the dried egg white protein formulation was 80 wt%, with the amount of pregelatinized starch being adjusted for variations in the protein content of the dried egg white protein formulation.

To assess the adequacy of the manufacturing process, blend uniformity was assessed for a representative batch prepared on a 7kg scale after the final mixing step. A sample of the formulation corresponding to 1 to 3 capsules was extracted from the prepared formulation at 10 different locations using a sampler. The protein content of each sample from the panel was determined using a bicinchoninic acid (BCA) assay. The results are summarized in table 11.

Table 11: blend uniformity results for representative batches

% LC: percentage of label declaration

0.2mg, 1mg, 6mg, 12mg and 300mg of the egg white protein formulations of the label claim were filled into hypromellose-based draw-off capsule shells to provide individual measured doses. For dose strengths of 0.2mg to 6mg, a #2 capsule shell of a size having a target powder fill weight of 180mg was selected. Capsule shell #00 with a target fill weight of 500mg was selected for higher dose strength to accommodate up to 300mg of ovalbumin with sufficient diluent, lubricant, and glidant to allow robust processing and deliverable mass. The egg white protein formulation is encapsulated in a capsule using a Bosch GKF701 encapsulator at a production rate of up to 700 capsules per minute. The formulation is fed into an enclosure and is tamped into the dosing tray using a series of tamping pins. The thickness of the dosing disc and the position of the packing pin can be adjusted to adjust the capsule filling weight. The empty capsule shells are opened by an enclosure, a slug of powder is inserted into the body of each opened capsule shell, and the capsule shells are then closed.

The deliverable mass percentage of the capsules was measured by sampling a group of ten prepared capsules at 0.2mg, 1mg, 6mg, 12mg and 300mg dose statements. The capsules were weighed, opened, and the formulation contained within the capsules was poured out. The mass of the formulation deliverable from the capsule is measured. Compressed air is then blown into the capsules to remove residual formulation and the empty capsules are weighed. The mass percent delivered and RSD for each dose statement are summarized in table 12.

Table 12: deliverable mass summary of representative batches

The content uniformity of the capsules was also determined by comparing the protein content in the deliverable mass to the nominal dose (i.e., label statement). Protein content was determined using BCA assay. The content uniformity of the multiple dose containers at different doses is summarized in table 13.

Table 13: summary of content uniformity for representative batches

Example 4: oral immunotherapy study for the treatment of egg allergy

A randomized, double-blind, placebo-controlled study of the efficacy and safety of pharmaceutical compositions containing raw dried egg white protein powder derived from chicken eggs and excipients in an oral immunotherapy regimen will be conducted. Placebo formulated with inactive compound and corresponding excipient will be used in the placebo group of this study. Figure 9 provides a schematic of the study.

Eligible subjects are (1) human individuals aged 4 to 26 years; (2) IgE-mediated egg allergy with physician diagnosis that develops an allergenic response within 2 hours of known oral exposure to eggs or egg-containing food; (3) has more than or equal to 7kU_ASerum ovalbumin specific IgE levels,/L; (4) development of dose-limiting allergy symptoms following single dose ≦ 300mg of dried ovalbumin protein administered in a double-blind placebo-controlled food challenge (DBPCFC); (5) for female individuals with fertility, reliable methods of birth control are used; and (6) are not otherwise excluded according to exclusion criteria.

Exclusion criteria included (1) a history of severe or life-threatening anaphylaxis or anaphylactic shock within 60 days prior to screening; (2) a 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) a history of mast cell disorders (e.g., systemic mastocytosis, urticaria pigmentosa, chronic idiopathic or chronic physical urticaria rather than simple artificial urticaria [ e.g., cold urticaria, urticaria cholinergic, or hereditary or idiopathic angioedema) ]; (4) mild or moderate asthma with uncontrolled or difficult control, or severe persistent asthma; (5) history of high dose corticosteroid drug use (e.g., prednisone or equivalent at 1mg/kg-2mg/kg >3 days); (6) a history of cardiovascular disease (including uncontrolled or inadequately controlled hypertension); (7) a history of chronic diseases (except asthma, atopic dermatitis or allergic rhinitis) that become unstable or require a change in a chronic treatment regimen or are at significant risk of becoming unstable or requiring a change in a chronic treatment regimen, including malignancies and autoimmune diseases within 5 years prior to screening; (8) a history of cardiovascular disease, including uncontrolled or inadequately controlled hypertension; (9) use of a beta-blocker (oral), angiotensin converting enzyme inhibitor, angiotensin receptor blocker, calcium channel blocker or tricyclic antidepressant; (10) antihistamines and other medications that may interfere with allergic reactions that assess 5 half-lives of the medication cannot be discontinued before the first day of screening and withdrawal from the Skin Prick Test (SPT) and food challenge and dose escalation; (11) lack of available palatable vehicle food to which the subject is not allergic; (12) hypersensitivity to wheat or oats; (13) hypersensitivity to epinephrine or any excipient in an epinephrine auto-injector; (14) using any therapeutic antibody or any immunomodulatory therapy (including immunosuppressive drugs (except aeroallergens or venom immunotherapy used during the maintenance phase within 6 months prior to screening)); (15) any type of egg or other food allergen immunotherapy is currently being received or received within 5 years prior to screening; (16) prior to screening, another clinical study was enrolled for 30 days or 5 half-lives (whichever is longer) of the study products; (17) during the establishment/up-regulation of immunotherapy for any non-egg allergen; and (18) pregnancy or current breast feeding.

The initial screen will include a dry egg white double-blind placebo-controlled food challenge (DBPCFC) and an open baked whole egg food challenge. DBPCFC is a procedure under medical supervision performed by: subjects, parents/caregivers, and study site workers, who were not aware of the random order of the challenge days, were fed dry egg white and placebo in measured, increasing doses over 2 separate days. The order of challenge is not known to the research site personnel until after the end of two days of DBPCFC. Food challenge materials will be prepared by designated non-blind personnel who are not involved in DBPCFC administration, monitoring or evaluation results. DBPCFC performed in this study will follow the guidelines for actual allergy (PRACTALL) for safety, assessment and scoring (Sampson et al, j. allergy clin. immunol., vol 130, p. 1260-. The DBPCFC program was performed during screening and at study exit. A summary of screening and withdrawal DBPCFC dosing schedules is provided in table 14 below.

Table 14: screening and withdrawal of DBPCFC challenge doses

The open baked whole egg food challenge is a procedure performed under medical supervision by feeding a test food product (a baked food product with eggs) at a measured, increased dose. The challenge to be performed under this study will follow a procedure that conforms to the PRACTALL guidelines for safety, assessment, and scoring. Open baking whole egg food challenges were performed during screening and at study exit. During the challenge, the subject will attempt to consume one whole muffin, according to the dosing schedule, where one muffin contains about one third of one whole egg, which is equivalent to about 2000mg egg protein. A summary of the screening and open baking whole egg food challenge dosing schedules is provided in table 15 below.

Table 15: screening and withdrawal of open baked whole egg food challenge dose

Eligible individuals were blinded with 2:1 randomized assignment of pharmaceutical compositions or placebo. Randomization will stratify the baseline reactivity of baked eggs in the open-baked whole egg food challenge at screening. During the course of the study, subjects who tolerated approximately 2000mg of accumulated baked egg protein (one muffin, which contained approximately one third of a whole egg) were allowed to consume the baked egg product. Subjects with dose-limiting allergy symptoms during the open-baked whole egg food challenge will be considered baked egg intolerant and will be instructed to avoid all forms of eggs during the study.

Subjects will begin initial dose escalation on day 1 under medical supervision at the study site and, where tolerated, step-wise dose escalation study product (up to 5 single doses of 0.2mg, 0.4mg, 0.8mg, 1.2mg and 2 mg) will be administered at intervals of 20 to 30 minutes. Subjects who tolerate a single dose of at least 1.2mg on day 1 will return on day 2 to receive a single confirmatory 1mg dose. Subjects who tolerate a validated 1mg dose without exceeding mild allergic symptoms that are not dose limiting will begin the up-dosing phase. Subjects who did not tolerate a 1.2mg dose on day 1 or a 1mg dose on day 2 will be discontinued early in the study.

Up-dosing will be about 6 months (22 weeks-40 weeks), with dose escalation occurring about every 2 weeks. The daily dose of the study product during the up-dosing period will be 1mg, 3mg, 6mg, 12mg, 20mg, 40mg, 80mg, 120mg, 160mg, 200mg, 240mg and 300 mg. The first dose of the study 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 with tolerance.

Subjects who reached a 300 mg/day dose within 40 weeks and tolerated the first 300mg dose without exceeding mild allergic symptoms which were not dose limiting will begin the maintenance phase. Subjects who did not reach the 300 mg/day dose within 40 weeks of day 1 will be discontinued early in the study.

Subjects who begin maintenance treatment will continue to administer study product at 300 mg/day for approximately 12 weeks per day, which may be extended by up to an additional 4 weeks to accommodate dose adjustments during the last 2 weeks of maintenance. Study site visits will occur approximately every 4 weeks. During a maintenance visit, the study product will be administered under medical supervision at the study site; subsequent maintenance doses will be administered daily at home with tolerance.

Depending on the adverse events or other complications experienced by the subject, dose adjustments will be allowed during the up-dosing phase or the maintenance phase as approved by the investigator. During the up-dosing phase or the maintenance phase, the dose may be adjusted in response to a dose-related allergic reaction according to one of the following: (1) administering the next dose of the study product under medical supervision at the study site; (2) delay study product dose escalation for an additional 1 to 2 weeks; (3) reducing the study product dose level by 1 or 2 dose levels; (4) temporary maintenance of the study product; or (5) discontinuing study product administration and discontinuing the subject early in the study. The severity of symptoms will guide the dose reduction of the study product for both acute and chronic or recurrent symptoms.

The dosage may also be adjusted for reasons other than the allergic reaction caused by the pharmaceutical composition, such as the onset of asthma or other atopic diseases, illness, or menstruation. The amount of dose reduction may range from 1 dose level (i.e., the previous dose level) to about 50% (rounded to the nearest feasible whole dose), at the discretion of the investigator. The minimum dose level is 1 mg. If the dose is reduced for reasons other than allergic reaction to the pharmaceutical composition, the reduced dose will be administered for 2 weeks and the subject will fully recover (i.e., baseline state) for at least 3 days, depending on the severity of the disease as assessed by the investigator, and then attempt a re-escalation of the dose at the study site. Dosing will be stopped and the subject will abort early if any of the following dose adjustment conditions are met: dose levels cannot be escalated after 3 consecutive failed attempts, with at least 2 weeks between each escalation attempt; or dose reduction could not be tolerated after 3 attempts to reduce dose levels.

At the end of maintenance, subjects will have a single highest challenge dose of up to 2000mg of dry egg white protein (cumulative 4043 or 4044mg) at the time of withdrawal from the DBPCFC, followed by an open-baked whole egg food challenge within 7 days after the second day of withdrawal from the DBPCFC. All subjects had to tolerate a 300mg daily dose of study product for at least 2 consecutive weeks before exiting the DBPCFC.

Subjects who completed the challenge of exiting the DBPCFC and open baking whole egg food will exit (complete) the study. After exiting the study and after all major data queries to the subject are resolved, the study treatment assignment will be non-blind to the subject. For subjects with early discontinuation of the study, study treatment assignment will be non-blinded after study completion.

The study endpoints of the study included (1) the proportion of subjects who tolerated a single highest dose of at least 300mg, at least 600mg, at least 1000mg or at least 2000mg of raw egg white protein with no more than mild allergic symptoms at the time of withdrawal from DBPCFC treated with the pharmaceutical composition compared to treatment with placebo; (2) a proportion of subjects who could not tolerate a cumulative dose of about 2000mg baked egg protein at screening and subsequently tolerated a cumulative dose of about 2000mg baked egg protein at study exit; and (3) summary of safety of treatment.

Example 5: oral immunotherapy study for the treatment of egg allergy

Eligible subjects are (1) human individuals aged 4 to 26 years; (2) IgE-mediated egg allergy with physician diagnosis that develops an allergenic response within 2 hours of known oral exposure to eggs or egg-containing food; (3) has more than or equal to 5kU_ASerum ovalbumin specific IgE levels,/L; (4) development of dose-limiting allergy symptoms following single dose ≦ 300mg of dried ovalbumin protein administered in a double-blind placebo-controlled food challenge (DBPCFC); (5) for female individuals with fertility, reliable methods of birth control are used; and (6) are not otherwise excluded according to exclusion criteria.

Table 16: screening and withdrawal of DBPCFC challenge doses

Table 17: screening and withdrawal of open baked whole egg food challenge dose

Subjects will begin initial dose escalation on day 1 under medical supervision at the study site and, where tolerated, step-wise dose escalation study product (up to 5 single doses of 0.2mg, 0.4mg, 0.8mg, 1.0mg and 2 mg) will be administered at intervals of 20 to 30 minutes. Subjects who tolerate a single dose of at least 1.0mg on day 1 will return on day 2 to receive a single confirmatory dose of 1.0 mg. Subjects who tolerate a validated 1.0mg dose without exceeding mild allergic symptoms that are not dose limiting will begin the up-dosing phase. Subjects who did not tolerate a 1.0mg dose on day 1 or a 1mg dose on day 2 will be discontinued early in the study.

Example 6: RP-HPLC determination

RP-HPLC methods for high resolution identification and protein profiling of dry egg white protein powders (drug substances) and pharmaceutical compositions of egg white proteins (drug products) were developed.

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

To prepare the pharmaceutical composition (i.e., the drug product) for assay, the volume of diluent depends on the dosage strength of the sample being tested. A specific dosage level of 10 capsules may be blended together and diluted with water to an egg white protein concentration of about 0.05 mg/ml. The diluted drug product sample may then be shaken on an orbital shaker for 30 minutes and diluted to a working concentration of about 0.038mg/ml to about 0.042mg/ml ovalbumin concentration. The samples can then be transferred to a 15ml conical centrifuge tube and centrifuged at 3,200rpm for 30 minutes. The clear supernatant can then be filtered through a 0.45 micron syringe filter, discarding the first 2 ml.

Using the modified RP-HPLC method, the major egg white protein components were well resolved with typical retention times of about 15.5 minutes (myoglobin), 16.1 minutes (carbonic anhydrase), 7.8 minutes (ovomucoid), 12.2 minutes (lysozyme), 14.4 minutes (ovotransferrin) and 18.1 minutes (ovalbumin). A sample RP-HPLC chromatogram of the pharmaceutical formulation using the modified RP-HPLC method is shown in figure 11. The analytical sample of the drug substance or drug product may be compared to a reference standard of a single protein (i.e., a purified allergen or other egg white protein) and/or a previously validated sample of the drug substance or drug product. The diluent of the formulated drug showed a broad retention curve with maximum amplitude after 22 minutes. The peak area for each individual protein can be calculated by subtracting the area attributable to the diluent from a reference standard of diluent and optionally any other individual excipients.

Example 7: stability study

Stability studies were performed with samples of pharmaceutical formulations of ovalbumin. Samples were tested under normal, accelerated and stress stability conditions, such as storage between 25 ℃ and 40 ℃.

In one experiment, 0.2mg capsules were stored in a high density polyethylene bottle along with a 1g desiccant sachet. The bottles were stored at 5 ℃. + -. 3 ℃, 25 ℃/60% RH, 30 ℃/65% RH or 40 ℃/75% RH.

At different time points, samples were assayed for appearance, deliverable mass, protein profile (by RP-HPLC), total protein (by BCA) and relative potency (by ELISA). The appearance was evaluated by opening individual capsules and placing a small amount of product on a clean watch glass. The material was observed for the presence of foreign matter on a white background. The deliverable quality was evaluated by: the method comprises weighing a complete capsule, emptying the capsule into a tared receptacle, 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 is delivered into the tared receptacle. Protein profiles were assessed by measuring the proportion of RP-HPLC peak areas attributable to Gal d 1, Gal d 2, Gal d 3 and Gal d 4. For earlier time points (between 1 and 6 months), the first RP-HPLC method described in example 1 was used. For later time points, the modified RP-HPLC method described in example 6 was used. Both RP-HPLC methods were used at the 6 month time point. Relative potency was assessed by ELISA and total protein by BCA as described in example 1. BCA is reported as a percentage of total egg white protein claimed by the tag.

The results of 0.2mg capsules after storage at 2 ℃ -8 ℃ are summarized in the following table. For the 6 month time point, both the older RP-HPLC method of example 1 and the modified RP-HPLC of example 6 were employed. The first value at the 6 month time point corresponds to the first RP-HPLC method and the second value at the 6 month time point corresponds to the modified RP-HPLC method.

The major allergen level and relative potency of the pharmaceutical formulation is stable between 0 and 12 months after storage between 2-8 ℃.

The results of 0.2mg capsules after accelerated stability storage conditions at 25 ℃/60% RH are summarized in the table below. For the 6 month time point, both the older RP-HPLC method and the modified RP-HPLC method described were employed. The first value at the 6 month time point corresponds to the first RP-HPLC method and the second value at the 6 month time point corresponds to the modified RP-HPLC method.

The major allergen levels and relative potency of the pharmaceutical formulations were stable between 0 and 12 months after 25 ℃/60% RH storage.

The results of 0.2mg capsules after accelerated stability storage conditions at 30 ℃/65% RH are summarized in the table below. For the 6 month time point, both the older RP-HPLC method and the modified RP-HPLC method described in the previous examples were employed, but only the older RP-HPLC values were reported for comparing the T ═ 0 time points.

The major allergen and relative potency of the pharmaceutical formulations are stable between 0 and 6 months after accelerated stability conditions of 30 ℃/65% RH storage.

The results of 0.2mg capsules after stress stable storage conditions at 40 ℃/75% RH are summarized in the table below. For the 6 month time points, both the older RP-HPLC method and the modified RP-HPLC method described in the previous examples were employed, but only the older RP-HPLC method values were reported to compare T ═ 0, 1, and 3 month time points.

The major allergen and relative potency of the pharmaceutical formulations were stable between 0 and 6 months even under stress stable storage conditions of 40 ℃/75% RH.

Stability studies were performed with a number of capsules containing 1mg, 6mg, 12mg and 300mg drug product doses. The results indicate that the pharmaceutical formulations are stable across dose levels under different storage conditions.

Example 8: oral immunotherapy of egg allergy in patients

Treating a patient with a diagnosed egg allergy with a pharmaceutical composition of egg white protein. The patient underwent an initial dose escalation, up-dosing phase, and maintenance phase according to an oral immunotherapy schedule. The indicated dose during the up-dosing phase or the maintenance phase may be adjusted if the subject experiences an adverse allergic response to a previous dose and/or experiences a complication associated with increased sensitivity to an allergen not associated with administration of a dose of the pharmaceutical formulation.

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

Example 9: low dose preparation of egg white protein formulations

Formulated pharmaceutical compositions for preparing low doses (including 0.2mg and 1mg doses) for oral immunotherapy can be prepared as follows. The starting drug substance was dried egg white protein powder. The dried egg white protein powder is characterized by comparison to a reference standard or a predefined acceptance standard to ensure consistent total protein levels and allergen levels, such as levels of Gal d 1, Gal d 2, Gal d 3 and Gal d 4. The relative potency of the egg white protein powder was also confirmed, e.g. by ELISA against ovomucin-like proteins and compared to predefined acceptance criteria.

The dry egg white protein powder is manually mixed with a first portion of pregelatinized starch to form a first mixture. The first mixture is then co-screened with a second portion of the pregelatinized starch through a mesh screen, and the co-screened material is then manually mixed to form a second mixture. The second mixture is then mixed with a third portion of the pregelatinized starch by a blender (e.g., a drum blender) to form a third mixture. The third mixture is then mixed with a fourth portion of the pregelatinized starch by a blender (e.g., a drum blender) to form a fourth mixture. The fourth mixture is then mixed with a fifth portion of the pregelatinized starch by a blender (e.g., a drum blender) to form a fifth mixture. The fifth mixture is then mixed with microcrystalline cellulose by a blender (e.g., a drum blender) and then further mixed by high shear mixing (e.g., in a cone mill) to form a sixth mixture. Separately, a sixth portion of pregelatinized starch and magnesium stearate are mixed together to form a seventh mixture, which is then passed through a mesh screen to screen the seventh mixture. The seventh mixture is then mixed with the sixth mixture in a blender (e.g., a drum blender) to form the egg white protein formulation.

Once formulated, the overall formulation is characterized by assessing total protein, content uniformity, individual allergen levels (such as levels of Gal d 1, Gal d 2, Gal d 3, and Gal d 4), relative potency of the allergen (such as by ELISA for ovomucoid), and water activity. Each evaluation is judged according to predetermined acceptance criteria. Once the monolithic formulation is validated, it is dispensed into a container, such as a capsule or sachet. Containers from the same production batch form a batch. A single container or multiple containers from the same batch are also evaluated. A validation batch comprising a plurality of containers is then dispensed for oral immunotherapy of egg allergy.

Example 10: low dose preparation of egg white protein formulations

Formulated pharmaceutical compositions for preparing low doses (including 3mg and 6mg doses) for oral immunotherapy can be prepared as follows. The starting drug substance was dried egg white protein powder. The dried egg white protein powder is characterized by comparison to a reference standard or a predefined acceptance standard to ensure consistent total protein levels and allergen levels, such as levels of Gal d 1, Gal d 2, Gal d 3 and Gal d 4. The relative potency of the egg white protein powder was also confirmed, e.g. by ELISA against ovomucin-like proteins and compared to predefined acceptance criteria.

The dry egg white protein powder is mixed with a first portion of pregelatinized starch to form a first mixture. The first mixture is mixed with the second portion of pregelatinized starch by high shear mixing (e.g., a cone mill), and then further mixed in a blender (such as a drum blender) to form a second mixture. The second mixture is then mixed with a third portion of the pregelatinized starch to form a third mixture. The third mixture is then mixed with microcrystalline cellulose in a blender (such as a drum blender) to form a fourth mixture, which is then further mixed by high shear mixing (e.g., in a cone mill). Separately, a fourth portion of the 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 an egg white protein preparation.

Example 11: moderate of egg white protein preparationDose preparation

Formulated pharmaceutical compositions for preparing intermediate doses (including 12mg doses) for oral immunotherapy may be prepared as follows. The starting drug substance was dried egg white protein powder. The dried egg white protein powder is characterized by comparison to a reference standard or a predefined acceptance standard to ensure consistent total protein levels and allergen levels, such as levels of Gal d 1, Gal d 2, Gal d 3 and Gal d 4. The relative potency of the egg white protein powder was also confirmed, e.g. by ELISA against ovomucin-like proteins and compared to predefined acceptance criteria.

The dry egg white protein powder is manually mixed with a first portion of pregelatinized starch and colloidal silica to form a first mixture. The first mixture is then mixed with a second portion of the pregelatinized starch using high shear mixing (e.g., in a cone mill) to form a second mixture, which is then further mixed in a blender (e.g., a drum blender) with lower shear. The second mixture is then mixed (using, for example, a blender, such as a drum blender) with a third portion of the pregelatinized starch to form a third mixture. The third mixture is then mixed with a fourth portion of the pregelatinized starch and microcrystalline cellulose in a blender (such as a drum blender) to form a fourth mixture, which is further mixed by high shear mixing (e.g., using a cone 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.

Example 12: intermediate dosage preparation of egg protein preparation

Formulated pharmaceutical compositions for preparing intermediate doses (including 20mg and 40mg doses) for oral immunotherapy may be prepared as follows. The starting drug substance was dried egg white protein powder. The dried egg white protein powder is characterized by comparison to a reference standard or a predefined acceptance standard to ensure consistent total protein levels and allergen levels, such as levels of Gal d 1, Gal d 2, Gal d 3, and Gal d 4. The relative potency of the egg white protein powder was also confirmed, e.g. by ELISA against ovomucin-like proteins and compared to predefined acceptance criteria.

The dry egg white protein powder is manually mixed with a first portion of pregelatinized starch and colloidal silica 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 cone mill) to form a second mixture, which is then further mixed in a blender (e.g., a drum blender). The second mixture is then mixed with a third portion of pregelatinized starch and microcrystalline cellulose in a blender (such as a drum blender) to form a third mixture, which is then further mixed by high shear mixing (e.g., in a cone mill). Separately, a fourth portion of the 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 an egg white protein preparation.

Example 13: high dose preparation of egg white protein formulations

Formulated pharmaceutical compositions for preparing high doses of oral immunotherapy (including 120mg, 160mg, 200mg, 240mg and 300mg doses) may be prepared as follows. The dried egg white protein powder is characterized by comparison to a reference standard or a predefined acceptance standard to ensure consistent total protein levels and allergen levels, such as levels of Gal d 1, Gal d 2, Gal d 3 and Gal d 4. The relative potency of the egg white protein powder was also confirmed, e.g. by ELISA against ovomucin-like proteins and compared to predefined acceptance criteria.

The dry egg white protein powder is manually mixed with a first portion of pregelatinized starch and colloidal silica to form a first mixture. The first mixture is then mixed with microcrystalline cellulose by high shear mixing (e.g., in a cone mill) to form a second mixture, which is then further mixed in a blender, such as a drum blender. Separately, a second portion of the 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 drum blender) to form an egg white protein formulation.

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Formulation for egg oral immunotherapy, preparation method and treatment for egg allergy

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