Methods for characterizing compositions comprising peanut antigens

Abstract

Methods for determining the in vitro release profile of peanut allergens in a sample are provided. Methods for determining one or more characteristics of peanut allergens in a sample are provided.

Description

[0001] This invention application is a divisional application based on an invention patent application filed on December 28, 2016, with application number 201680082864.X (international application number PCT / IB2016 / 001944), entitled “Method for Characterizing a Composition Containing Peanut Antigens”.

[0002] Related applications

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 272,094, filed December 29, 2015, and French Patent Application No. 163306642.6, filed December 8, 2016, each of which is incorporated herein by reference in its entirety. Field of the Invention

[0004] The present invention relates to methods for characterizing, e.g., determining the release profile, allergen signature, etc., of a therapeutic composition comprising a peanut allergen for treating, alleviating, or otherwise reducing peanut allergy in a subject. Background Art

[0005] Peanut allergy develops when the body's immune system produces an abnormal hypersensitivity reaction to one or more peanut allergens. Peanut allergy is one of the most common food allergies in both children and adults. It is of particular concern because it is relatively common, often a lifelong condition, and can cause severe allergic reactions. Peanut allergy is the leading cause of anaphylaxis and death from food allergies. Peanuts are a common food ingredient, making strict avoidance difficult. Consequently, the rate of accidental peanut ingestion is relatively high for those who try to avoid them. For these reasons, peanut allergy has become a significant public health issue.

[0006] Current research is focused on developing compositions for the treatment of peanut allergy. Methods are needed to determine in vitro peanut allergen release data for known and newly developed compositions, both for quality control and for predicting in vivo release profiles. Summary of the Invention

[0007] The present invention is based, in part, on the discovery of highly sensitive methods for determining the release profile of allergens and / or the allergenic characteristics of compositions containing allergens. The methods described herein provide the sensitivity and accuracy necessary to obtain a profile of peanut allergens present in a sample, such as a protein extract, a therapeutic composition, or a dissolution or release medium, and allow for the measurement of low-level, relevant allergens present in the sample. In an exemplary embodiment, the methods involve detecting allergen digestion products present in the form of one or more isoforms of one or more allergens in the sample. Identification of allergen digestion products present in the form of multiple isoforms of peanut allergens provides qualitative and quantitative information about the sample, which can serve as an indicator of batch-to-batch consistency and can provide release profiles of allergens stored on or in a matrix, such as nanoparticles.

[0008] In one aspect, the invention features a highly sensitive method for determining the signature of a peanut allergen in a composition. The method includes the steps of digesting a peanut allergen present in the composition from the composition (e.g., a medium or other sample) to produce an allergen digestion product, fragmenting the allergen digestion product to produce peptide fragments, detecting and identifying the allergen digestion product by mass spectrometry, and determining the signature of the peanut allergen in the composition. In certain embodiments, the method is used to determine the signature of one or more peanut allergens present at low concentrations in a composition. The signature includes the type and amount (e.g., relative amount) of the allergen in the composition.

[0009] In certain embodiments, the composition is an aqueous medium, such as an aqueous pharmaceutical composition, an analytical sample, a dissolution medium, or a release medium. In some embodiments, the total amount of peanut allergen in the composition is very low. For example, the amount of a particular peanut allergen (e.g., Ara h1, Ara h2, Ara h3, or Ara h6) can be less than about 2 μg / ml, less than about 1.5 μg / ml, less than about 1 μg / ml, or less than about 0.5 μg / ml, such as about 0.2 μg / ml.

[0010] In some embodiments, the method further comprises the step of comparing the peanut allergen signature to a signature standard. The signature standard can be a peptide spectrum set by a regulatory agency such as the FDA (Food and Drug Administration) or the EMA (European Medicines Agency), a peptide spectrum established according to industry standards, or an expected profile determined through repeated experiments. The signature standard can specify the types and relative amounts of peanut allergens expected to be found in the sample.

[0011] In certain exemplary embodiments, the allergen digest product is from about 4 amino acids to about 50 amino acids in length, from about 6 amino acids to about 30 amino acids in length, or from about 15 amino acids to about 20 amino acids in length, or about 15, 16, 17, 18, 19, or 20 amino acids in length.

[0012] In certain exemplary embodiments, the steps of fragmenting the allergen digestion products and detecting the peptide fragments are performed by a method comprising tandem mass spectrometry, such as liquid chromatography-tandem mass spectrometry (LC-MS-MS), nanotandem mass spectrometry (nanoLC-MS-MS), or nanohigh performance liquid chromatography-tandem mass spectrometry (nanoHPLC-MS-MS).

[0013] In certain exemplary embodiments, the signature (e.g., peanut allergen signature and / or signature standard) comprises one or more Ara h 1 allergen digestion products having an amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 70, SEQ ID NO: 177, SEQ ID NO: 155, SEQ ID NO: 93, and SEQ ID NO: 40. In some embodiments, the signature comprises allergen digestion products from Ara h 1, Ara h 2, and Ara h 6. In other embodiments, the signature comprises allergen digestion products from Ara h 1, Ara h 2, Ara h 3, and Ara h 6. In some embodiments, the signature comprises one or more allergen digestion products having an amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 70, SEQ ID NO: 177, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 236, and SEQ ID NO: 237. In some embodiments, the feature comprises one or more allergen digestion products having an amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 70, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 236, and SEQ ID NO: 237.

[0014] In some embodiments, allergen digestion products suitable for use in establishing a peanut allergen profile are present in more than one isoform of a peanut allergen, such as 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, or more isoforms of a particular allergen (e.g., an Ara h protein or polypeptide). For example, allergen digestion products suitable for use in a peanut allergen profile may be present in one or any combination of the following: 2, 3, 4, 5, 6, 7, 8, or more isoforms of Ara h 1, 2, 3, 4, 5, 6, 7, 8, or more isoforms of Ara h 2, 2, 3, 4, 5, 6, 7, 8, or more isoforms of Ara h 3, and / or 2, 3, 4, 5, 6, 7, 8, or more isoforms of Ara h 6. In some embodiments, allergen digestion products suitable for use in a peanut allergen profile are present in all isoforms of one or more peanut allergens.

[0015] In certain exemplary embodiments, the peanut allergen is digested with one or more proteases. For example, the peanut allergen can be digested with one or more proteases selected from the group consisting of trypsin, endoproteinase Lys-C, and endoproteinase Arg-C.

[0016] In certain exemplary embodiments, the composition comprising peanut allergens further comprises an internal standard. For example, in some embodiments, the internal standard comprises one or more heavy isotopes, such as 13 C and / or 15 N. In certain embodiments, the internal standard is not a full-length allergen, but rather a fragment of a peptide. The fragment is typically less than 50 amino acids in length (such as, for example, about 4 to about 50 amino acids in length, about 6 to about 30 amino acids in length, or about 10 to about 20 amino acids in length). In some embodiments, the fragment has the sequence of an expected or predicted allergen digestion product. In one embodiment, the internal standard consists of a plurality of peanut allergen peptides, each of which is labeled at the C-terminus with a heavy isotope. For example, an exemplary internal standard may include any combination of two or more isotopically labeled fragments of an Ara h1 peptide, two or more isotopically labeled fragments of an Ara h2 peptide, two or more isotopically labeled fragments of an Ara h3 peptide, and two or more isotopically labeled fragments of an Ara h6 peptide. In one embodiment, a composition comprising a peanut antigen is digested with trypsin to remove the peanut antigen. 13 C and / or 15 A standard mixture of N-labeled peanut allergen peptides is added to the digested mixture and the composition is then determined by fragmentation, for example, by mass spectrometry, such as LC-MS or LC-MS-MS.

[0017] In certain exemplary embodiments, the signature comprises an allergen digest product that is free of missed proteolytic cleavage sites.

[0018] In another aspect, the invention features a method for determining a release profile (e.g., an in vitro release profile) of a composition comprising one or more peanut allergens. In one embodiment, the method comprises obtaining one or more samples from the composition at each of a plurality of time points, digesting peanut allergens present in the one or more samples to produce allergen digestion products, fragmenting the allergen digestion products to produce peptide fragments, and detecting the peptide fragments at at least two of the plurality of time points to determine a release profile of the peanut allergens. The composition can be, for example, a peanut extract, a therapeutic composition comprising a peanut allergen, a dissolution medium, or an analytical sample. In some embodiments, the composition is an aqueous medium. In some embodiments, the amount of peanut allergen in the composition is very low. For example, the amount of a particular peanut allergen (e.g., Ara h1, Ara h2, Ara h3, or Ara h6) can be less than about 2 μg / ml, less than about 1.5 μg / ml, less than about 1 μg / ml, or less than about 0.5 μg / ml, for example, about 0.2 μg / ml, or be about 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the allergens in a sample taken from the composition are digested to produce allergen digestion products that are between about 4 amino acids and about 50 amino acids in length, between about 6 amino acids and about 30 amino acids in length, or between about 15 amino acids and about 20 amino acids in length. The pattern of allergen digestion products obtained after digestion creates a peptide signature for the sample.

[0019] In some embodiments, the steps of fragmenting the allergen digest and detecting the peptide fragments comprise one or more of a separation method and a peptide detection method. For example, in some embodiments, the steps of detecting and identifying the peptide fragments comprise performing a method such as LC-MS-MS, nano-LC-MS-MS, HPLC-MS-MS, or nanoHPLC-MS-MS. In some embodiments, the peptide signature comprises a collection of fragments from one or more of the peanut proteins Ara h1, Ara h2, Ara h3, Ara h4, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h12, and Ara h13.

[0020] In some embodiments, the peptide signature comprises one or more Ara h 1 allergen digest products having an amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 70, SEQ ID NO: 177, SEQ ID NO: 155, SEQ ID NO: 93, and SEQ ID NO: 40.

[0021] In some embodiments, the peptide signature comprises one or more allergen digestion products from any combination of Ara h 1, Ara h2, and Ara h6, or one or more allergen digestion products from any combination of Ara h 1, Ara h2, Ara h3, and Ara h6.

[0022] In some embodiments, the signature comprises one or more allergen digestion products having an amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 70, SEQ ID NO: 177, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 236, and SEQ ID NO: 237.

[0023] In certain embodiments, the allergen digestion product is present in more than one isoform of peanut allergen, such as one, two, three, four or all isoforms of peanut allergen, such as one, two, three, four or all isoforms of Ara h 1. In one embodiment, the allergen digestion product is in more than one isoform of peanut allergen.

[0024] In certain exemplary embodiments, the peanut allergen is treated with one or more proteases, such as one or more proteases selected from the group consisting of trypsin, endoproteinase Lys-C, and endoproteinase Arg-C.

[0025] In certain embodiments, the sample, such as a sample taken from an extract or pharmaceutical composition, further comprises an internal standard. The internal standard is typically a peptide having the sequence of an expected or predicted allergen digestion product and is typically less than about 50 amino acids in length (e.g., about 4 to about 50 amino acids in length, about 6 to about 30 amino acids in length, or about 10 to about 20 amino acids in length, or about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acids in length). The internal standard can be added to the composition prior to sampling and / or added to the sample prior to the enzymatic digestion step. In certain exemplary embodiments, the internal standard comprises one or more heavy isotopes, such as, for example, 13 C and / or 15N.

[0026] In some embodiments, the signature comprises allergen digestion products that are free of missed (uncleaved) proteolytic cleavage sites.

[0027] In some embodiments, the composition comprises particles encapsulating the allergen and / or having the allergen bound to the surface. The particles may be, for example, nanoparticles, microparticles, films, capsules or hydrogels.

[0028] In some embodiments, a release profile is obtained over a period of time, such as, for example, a period of hours, for example, a three hour period, a six hour period, a twelve hour period, or a twenty-four hour period.

[0029] Specifically, the present invention relates to the following:

[0030] 1. A method for characterizing peanut allergens in an aqueous medium, comprising:

[0031] digesting peanut allergens present in an aqueous medium to produce allergen digestion products;

[0032] fragmenting the allergen digestion products to produce peptide fragments, and

[0033] The allergen digestion products of the peanut allergen in the aqueous medium are characterized.

[0034] 2. The method of claim 1 , wherein the aqueous medium is a dissolution or release medium.

[0035] 3. The method of claim 1 , wherein the aqueous medium is an analytical sample.

[0036] 4. The method of claim 1 , wherein the amount of peanut allergen in the medium is less than about 2 μg / ml, less than about 1.5 μg / ml, less than about 1 μg / ml, or less than about 0.5 μg / ml.

[0037] 5. The method of claim 1, further comprising the step of comparing the feature with a feature standard.

[0038] 6. The method of claim 1 , wherein the allergen digest product is about 4 amino acids to about 50 amino acids in length.

[0039] 7. The method of claim 1 , wherein the allergen digest product is about 6 amino acids to about 30 amino acids in length.

[0040] 8. The method of claim 1 , wherein the allergen digest product is about 15 amino acids to about 20 amino acids in length.

[0041] 9. The method of claim 1 , wherein the steps of fragmenting the allergen digestion products and determining the characteristics are performed by a method selected from one or any combination of LC-MS, LC-MS-MS, nano-LC-MS-MS and nanoHPLC-MS-MS.

[0042] 10. The method of claim 1 , wherein the signature comprises an Ara h 1 allergen digest product having an amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 70, SEQ ID NO: 177, SEQ ID NO: 155, SEQ ID NO: 93, and SEQ ID NO: 40.

[0043] 11. The method of claim 1 , wherein the signature comprises allergen digests from Ara h1, Ara h2, and Ara h6.

[0044] 12. The method of claim 1 , wherein the signature comprises allergen digests from Ara h1, Ara h2, Ara h3, and Ara h6.

[0045] 13. The method of claim 12, wherein the feature comprises an allergen digest product having an amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 70, SEQ ID NO: 177, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 236 and SEQ ID NO: 237.

[0046] 14. The method of claim 1, wherein the allergen digestion products are present in most isoforms of Ara h 1.

[0047] 15. The method of claim 1, wherein the allergen digestion products are present in at least 90% of the isoforms of Ara h 1.

[0048] 16. The method of claim 1, wherein the allergen digestion products are present in all isoforms of Ara h 1.

[0049] 17. The method of claim 1, wherein the allergen digestion products are present in most isoforms of Ara h 1, Ara h 2 and Ara h 6.

[0050] 18. The method of claim 1, wherein the allergen digestion products are present in most isoforms of Ara h1, Ara h2, Ara h3 and Ara h6.

[0051] 19. The method of claim 1, wherein the peanut allergen is digested using one or more proteases selected from the group consisting of trypsin, endoproteinase Lys-C, and endoproteinase Arg-C.

[0052] 20. The method of claim 1 , wherein the aqueous medium further comprises an internal standard.

[0053] 21. The method of claim 1 , wherein the internal standard comprises one or more heavy isotopes.

[0054] 22. The method of claim 1 , wherein the signature comprises allergen digestion products that are free of missed proteolytic cleavage sites.

[0055] 23. A method for determining an in vitro release profile of an aqueous medium containing a peanut allergen, comprising:

[0056] obtaining a sample from the aqueous medium at each of a plurality of time points;

[0057] digesting peanut allergens present in the sample to produce an allergen digestion product;

[0058] fragmenting the allergen digestion products to produce peptide fragments, and

[0059] The peptide fragments are detected at at least two of the plurality of time points to identify the allergen digestion products, thereby determining an in vitro release profile of the peanut allergen from the aqueous medium.

[0060] 24. The method of claim 23, wherein the aqueous medium is a dissolution or release medium.

[0061] 25. The method of claim 23, wherein the amount of peanut allergen in the aqueous medium is less than about 2 μg / ml, less than about 1.5 μg / ml, less than about 1 μg / ml, or less than about 0.5 μg / ml.

[0062] 26. The method of claim 23, wherein the aqueous medium is an analytical sample.

[0063] 27. The method of claim 23, wherein the allergen digest product is about 4 amino acids to about 50 amino acids in length.

[0064] 28. The method of claim 23, wherein the allergen digestion product is about 6 amino acids to about 30 amino acids in length.

[0065] 29. The method of claim 23, wherein the allergen digest product is about 15 amino acids to about 20 amino acids in length.

[0066] 30. The method of claim 23, wherein the steps of fragmenting the allergen digestion products and detecting the peptide fragments are performed by one or a combination of methods selected from the group consisting of chromatography-tandem mass spectrometry (LC-MS-MS), nanoLC-MS-MS, and nano high performance liquid chromatography-tandem mass spectrometry (nanoHPLC-MS-MS).

[0067] 31. The method of claim 23, wherein the signature comprises an Ara h 1 allergen digest product having an amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 70, SEQ ID NO: 177, SEQ ID NO: 155, SEQ ID NO: 93, and SEQ ID NO: 40.

[0068] 32. The method of claim 23, wherein the signature comprises allergen digests from Ara h 1, Ara h 2, and Ara h 6.

[0069] 33. The method of claim 23, wherein the signature comprises allergen digests from Ara h1, Ara h2, Ara h3, and Ara h6.

[0070] 34. The method of claim 23 , wherein the feature comprises an allergen digest product having an amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 70, SEQ ID NO: 177, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 236, and SEQ ID NO: 237.

[0071] 35. The method of claim 23, wherein the allergen digestion products are present in most isoforms of Arah1.

[0072] 36. The method of claim 23, wherein the allergen digestion products are present in at least 90% of the isoforms of Ara h 1.

[0073] 37. The method of claim 23, wherein the allergen digestion products are present in all isoforms of Ara h 1.

[0074] 38. The method of claim 23, wherein the allergen digestion products are present in most isoforms of Ara h 1, Ara h2, and Ara h6.

[0075] 39. The method of claim 23, wherein the peanut allergen is digested using one or more proteases selected from the group consisting of trypsin, endoproteinase Lys-C, and endoproteinase Arg-C.

[0076] 40. The method of claim 23, wherein said aqueous medium further comprises the use of an internal standard.

[0077] 41. The method of claim 40, wherein the internal standard comprises one or more heavy isotopes.

[0078] 42. The method of claim 23, wherein the features comprise allergen digest fragments that do not contain omitted proteolytic cleavage sites.

[0079] 43. The method of claim 23, wherein the composition comprises one or both of nanoparticles and microparticles.

[0080] 44. The method of claim 23, wherein the release profile is obtained over a period of three hours.

[0081] 45. The method of claim 23, wherein the release profile is obtained over a period of six hours.

[0082] 46. ​​The method of claim 23, wherein the release profile is obtained over a period of twelve hours.

[0083] 47. The method of claim 23, wherein the release profile is obtained over a period of twenty-four hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings.

[0085] Figures 1A-1C Depicted are high-quality Ara h 1 peptides (SEQ ID NO: 70) identified by trypsin digestion followed by MS-MS. (A) Depicts the sequence, the number of uncut trypsin cleavage sites ("missed"), the percentage of false discovery rate (FDR), the number of post-translational modification sites ("PTM"), confirmation that the identified sequence is unique to the target protein in BLAST (Basic Local Alignment Tool) ("BLAST"), the database searched, and the number of isoforms in the database containing the identified sequence. (B) and (C) depict the MS-MS data in tabular and graphical form, respectively. Detailed Description of the Invention

[0086] The present invention is based, in part, on the development of a sensitive analytical method for measuring and profiling the presence of peanut allergens in compositions (e.g., extracts or therapeutic compositions), and for determining release profiles, e.g., in vitro release profiles, of formulated compositions containing peanut allergens. The method involves detecting peptides present in one or more isoforms of one or more allergens in a sample. Identification of peptides found in multiple isoforms of peanut allergens provides qualitative and quantitative information about the sample, which can be an indicator of batch-to-batch consistency and can provide release profiles for antigens stored on or in a substrate, such as nanoparticles. The method is particularly suitable for analyzing the allergen content of compositions containing very low amounts of peanut allergen.

[0087] As used herein, the terms "peanut," "groundnut," and "Arachis hypogea" are used interchangeably and refer to a plant belonging to the Leguminosae family and the subfamily Papillionacea. More than 17 different peanut allergens have been identified. Peanut protein allergens include Ara h1, Ara h2, Ara h3, Ara h4, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h12, Ara h13, Ara h14, Ara h15, Ara h16, and Ara h17.GenBank accession numbers for cDNA sequences of exemplary allergens include L34402.1 (Ara h1), AY007229.1 (Ara h2.0101), AY158467.1 (Ara h2.0201), AF093541.1 (Ara h3.0101), AF086821.1 (Ara h3.0201), AF059616 (Ara h5), AF092846.1 (Ara h6), AF091737.1 (Ara h7), EU046325.1 (AraH7.0201), AY328088.1 (Ara h8.0101), EF436550.1 (Ara h8.0201), EU159429.1 (Ara h9.0101), and EU161278.1 (Ara h10.0101). h9.0201), AY722694.2(Ara h10.0101), AY722695.1(Ara h10.0201), DQ097716.1(Ara h11), EY396089.1(Ara h12), EY396019.1(AraH13), AAK13449(Ara h14.0101), AAK13450 (Ara h14.0102), AAT11925 (Arah14.0103), AAU21501 (Ara h15.0101) (see, for example, Leon et al., The peanut allergyepidemic: allergen molecular characterization and prospects for specific therapy. Expert Rev. Mol. Med. Vol. 9, Issue 1,January 2007; see also Arkwright et al., IgE Sensitization to the Nonspecific Lipid-Transfer Protein Ara h 9 and Peanut-Associated Bronchospasm, BioMed Research International, vol. 2013, Article ID 746507; see url address allergen.org / search.php?allergensource=Arachis+hypogaea).

[0088] In an exemplary embodiment, a profile of allergen digestion products in a sample is obtained by digesting peanut allergens present in the sample to produce allergen digestion products, fragmenting the allergen digestion products to produce peptide fragments, and detecting and identifying the peptide fragments to obtain a profile of the allergen digestion products. As used herein, "allergen digestion products" refers to peanut allergens present in a digested extract or sample, for example, using an enzyme (e.g., trypsin, endoproteinase Lys-C, endoproteinase Arg-C, etc.) to produce products that are smaller than the intact allergen. The term "allergen digestion product" also refers to the amino acid sequence of a peptide that would be produced if the peanut allergen were enzymatically digested.

[0089] As used herein, "allergen" refers to a subset of antigens (eg, peanut peptide antigens) that elicit the production of IgE in addition to other isotypes of antibodies. The terms "allergen," "natural allergen," and "wild-type allergen" are used interchangeably.

[0090] As used herein, "antigen" refers to a molecule (eg, peanut peptide) that elicits an antibody response (ie, a humoral response) and / or an antigen-specific reaction with T cells (ie, a cellular response) in an animal.

[0091] Non-limiting examples of enzymes, particularly proteases, suitable for digesting peanut allergens to produce allergen digestion products include, but are not limited to, trypsin, endoproteinase Glu-C, endoproteinase Asp-N, chymotrypsin, endoproteinase Lys-C and endoproteinase Arg-C, pepsin, papain, thermolysin, subtilisin, proteinase K, bromelain, thiol-specific protease (ficin), and the like.

[0092] As used herein, "peptide fragments" or "gas-phase peptide fragments" refer to any portion or part of an allergen that is less than the intact native allergen and is produced by a fragmentation process that does not utilize enzymes. Typically, fragmentation conditions are introduced in the gas phase, such as during a mass spectrometry step. In certain exemplary embodiments, the peptide fragments are less than 50 amino acids in length, e.g., from about 2 to about 50 amino acids in length, from about 6 to about 30 amino acids in length, or from about 15 to about 20 amino acids in length, or any value or subrange within these ranges. In certain exemplary embodiments, the peptide fragment is about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, or about 49 amino acids in length.

[0093] In certain exemplary embodiments, peptide fragments are generated by fragmenting the allergen digestion products and then using a separation process and peptide identification method, such as liquid chromatography-tandem mass spectrometry (LC-MS-MS), nano-LC-MS-MS, high performance liquid chromatography-tandem MS (HPLC-MS-MS), nanoHPLC-MS-MS, ultra high performance tandem MS (UPLC-MS-MS), nanoUPLC-MS-MS and ultra high performance tandem MS (UHPLC-MS-MS), nanoUHPLC-MS-MS, etc.

[0094] According to certain exemplary embodiments, the methods described herein further comprise performing mass analysis on the allergen digestion products and / or peptide fragments using a mass analyzer. The mass analyzer typically comprises a triple quadrupole mass analyzer. According to other embodiments, the mass analyzer may comprise a mass analyzer selected from the group consisting of: (i) a triple quadrupole mass spectrometer, (ii) an orbitrap, such as a Fourier transform orbitrap, such as an Orbitrap ELITE TM(Thermo Scientific); (iii) Fourier transform ("FT") mass analyzers; (ii) Fourier transform ion cyclotron resonance ("FTICR") mass analyzers; (iii) time-of-flight ("TOF") mass analyzers; (iv) orthogonal acceleration time-of-flight ("oaTOF") mass analyzers; (v) axial acceleration time-of-flight mass analyzers; (vi) magnetic sector mass spectrometers; (vii) Paul or 3D quadrupole mass analyzers; (viii) 2D or linear quadrupole mass analyzers; (ix) Penning trap mass analyzers; (x) ion trap mass analyzers; and (xiii) electrostatic Fourier transform mass spectrometers.

[0095] In certain exemplary embodiments, the methods described herein utilize a separation method, such as chromatography, e.g., liquid chromatography. According to one embodiment, the allergen digest is fragmented and / or the peptide fragments are detected and identified by: (i) high performance liquid chromatography ("HPLC"), (ii) anion exchange, (iii) anion exchange chromatography; (iv) cation exchange; (v) cation exchange chromatography; (vi) ion pair reversed phase chromatography; (vii) chromatography; (viii) unidimensional electrophoresis; (ix) multidimensional electrophoresis; (x) size exclusion; (xi) affinity; (xii) reversed phase chromatography; (xiii) capillary electrophoresis chromatography ("CEC"); (xiv) electrophoresis; (xv) ion mobility separation; (xvi) field asymmetric ion mobility separation or spectrometry ("FAIMS"); (xvii) capillary electrophoresis; and (xviii) supercritical fluid chromatography.

[0096] According to certain exemplary embodiments, the method further comprises ionizing the allergen digestion products and / or peptide fragments in the sample to be analyzed. The ion source may comprise a continuous ion source. According to one embodiment, the ion source may be selected from the group consisting of: (i) an electrospray ionization ("ESI") ion source; (ii) a matrix-assisted laser desorption ionization ("MALDI") ion source; (iii) a desorption ionization on silicon ("DIOS") ion source; and (iv) a desorption electrospray ionization ("DESI") ion source.

[0097] Peanut allergen signatures described herein are typically determined by measuring multiple reaction monitoring (MRM) transitions consisting of a peptide precursor ion, one or more fragment ions, and retention times. For example, such measurements are performed on a triple quadrupole instrument. Signatures can also be obtained by combining retention times of intact peptides with accurate high-resolution mass spectrometry. These quantitative methods typically require labeled internal standards and external synthetic peptide calibration curves. In one embodiment, signatures are obtained by nano-LC / MS / MS (nLC-MS-MS) analysis, where as many peptides as possible are fragmented and identified by database analysis and nLC / MS / MS datasets consisting of retention times, mass / charge values, and intensities, which are measured and compared to determine changes in the global proteome.

[0098] In certain exemplary embodiments, an internal standard is used that includes a combination of two or more allergen digestion products from a protein allergen, such as one or more of Ara h1, Ara h2, Ara h3, Ara h4, Ara h5, Ara h6, Arah7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h12, Ara h13, Arah14, Arah15, Ara h16, and Arah17. Internal standards typically have known peptide sequences and are provided in known quantities. In some embodiments, the internal standard includes a combination of allergen digestion products from Ara h1, Ara h2, and Ara h6 of the peanut allergen. In other embodiments, an internal standard is used that includes a combination of allergen digestion products from Ara h1, Ara h2, Ara h3, and Ara h6. In certain exemplary embodiments, a sample may include one or more internal standards comprising one or any combination of the allergen digestion products listed in Tables 19-23. In some embodiments, the standards are labeled, for example, with one or more heavy isotopes, e.g. 13 C or 15 N.

[0099] In certain embodiments, the allergen digest products selected for characterizing the composition uniquely represent a peanut allergen or family of peanut allergen isoforms and are present in most or all isoforms of the allergen from which they are derived, enabling their use for specific quantification of the allergen of interest, such as peanut allergens. Thus, in certain exemplary embodiments, a signature of allergen digest products is generated. As used herein, a "signature" or "allergy signature" refers to the presence of a specific combination and amount of a particular peanut allergen digest product (e.g., Ara h1, Ara h2, Ara h6, and / or Ara h3 allergen digest products). In certain exemplary embodiments, the signature comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more different allergen digestion products, each different product having a unique sequence.

[0100] The selection of peptides for peanut allergen characterization can be based on the identification of isoforms, including peptides identified by the digestion and fragmentation steps. Various sequence databases can be used to identify sequences of peanut allergen isoforms, including UniProt (online at uniprot.org, which includes the Swiss-Prot and TrEMBL databases), the Allergen Nomenclature database (online at allergen.org), GenBank (online at ncbi.nlm.nih.gov), GeneCards (online at genecards.org), Ensembl (online at ensemble.org), etc.

[0101] Any sequence alignment algorithm can be used to identify isoforms, including peptides identified after digestion and fragmentation of samples containing peanut allergens. Exemplary sequence alignment algorithms include BLAST (online at blast.ncbi.nlm.nih.gov / Blast.cgi), Clustal Omega (online at ebi.ac.uk / Tools / msa / clustalo), and the like.

[0102] Peanut allergen analysis methods can be used for a variety of purposes. For example, the methods described herein can be used to assess batch-to-batch reproducibility in peanut-containing compositions. The methods described herein can also be used to measure the release or leakage of peanut allergens from the surface or interior of a container or matrix, such as beads or particles (e.g., nanoparticles or microparticles), capsules, films or strips (e.g., for sublingual administration of a composition), or gels (e.g., hydrogels).

[0103] In some embodiments, the peptide signature comprises peptides comprising one or more immunogenic epitopes or one or more immunodominant epitopes of one or more peanut allergens. The peptide signature comprising immunogenic epitopes or immunodominant epitopes can provide a criterion for the immunogenicity of a composition comprising a peanut allergen.

[0104] As used herein, "sample" refers to any composition containing peanut allergens. Exemplary samples include, but are not limited to, peanut-containing extracts, peanut-containing powders, analytical samples containing one or more peanut allergens, pharmaceutical compositions (e.g., therapeutic vaccines), dissolution or release media, and the like. In typical embodiments, the sample will be aqueous.

[0105] The sample used in the allergen analysis method characterized by the present invention can be a peanut extract, such as an extract made from whole roasted or raw peanuts or from peanut flour. The extract can be used in a pharmaceutical composition, for example, for treating or preventing peanut allergies. The extract can be used in a composition for oral immunotherapy (OIT) or sublingual immunotherapy (SLIT), or in a composition for a nanoparticle composition, with or without an adjuvant. By evaluating the peanut allergen profile of the peanut extract, batch-to-batch consistency can be monitored. The peanut allergen profile can also be used as a factor in inferring the immunogenicity of the extract, as extracts with similar profiles are expected to have similar immunogenicity.

[0106] In some embodiments, the amount of peanut allergen in the composition is very low. For example, the amount of a particular peanut allergen (e.g., Ara h1, Ara h2, Ara h3, and / or Ara h6) can be less than about 2 μg / ml, less than about 1.5 μg / ml, less than about 1 μg / ml, or less than about 0.5 μg / ml, such as about 0.2 μg / ml.

[0107] Peanut protein extracts can be prepared by methods known in the art, including defatting and / or filtration methods, to produce peanut allergen preparations.

[0108] The sample used in the allergen analysis method described herein may be a therapeutic composition, such as a liquid formulation for administration orally, sublingually, mucosally, intradermally, subcutaneously, intravenously, intramuscularly, parenterally or by inhalation.

[0109] In some embodiments, the therapeutic composition will be in the form of a membrane or strip.Prior to analysis by the allergen analysis methods described herein, a sample may include a piece of membrane dissolved in a buffer.

[0110] In other embodiments, the sample may comprise a matrix, such as a nanoparticle, a capsule, a film or a tablet, or a gel, such as a hydrogel. The allergen analysis methods described herein can be used to measure the release of peanut allergen from a matrix, such as, for example, a nanoparticle or a capsule, or from a film or a hydrogel. Release can be from within the matrix, such as a particle, or from the exterior of the matrix (e.g., the surface). In one embodiment, the release profile is determined by performing a complete release of the peanut allergen and then measuring the controlled release, for example, over a period of time or under different culture or solution conditions (e.g., at different temperatures, pH, or the like). The amount of allergen released in the controlled release assay is typically reported as a fraction or percentage compared to the amount of allergen released under complete release conditions.

[0111] In certain exemplary embodiments, two or more features obtained at specific time points can be used to determine an in vitro release profile of a peanut allergen from a sample containing a matrix (e.g., an aqueous sample, e.g., a dissolution or release medium). The in vitro release profile can be determined over a period of hours, days, or weeks. In certain exemplary embodiments, the release profile is obtained over a period of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or about 24 hours. In certain exemplary embodiments, the release profile is obtained over a period of about 1, 2, 3, 4, 5, 6, or about 7 days. In certain exemplary embodiments, the release profile is obtained over a period of about one week, about two weeks, about three weeks, about four weeks, about five weeks, about six weeks, about seven weeks, or about eight weeks. In certain exemplary embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, or more features are obtained over a specific period of time. For example, the release burst of a peanut allergen can be assessed at evenly spaced time points over the first 24 hours with sufficient data to determine the burst kinetics, and then the remaining amount of the same allergen can be released from the sample to help assess the overall product composition performance. The release profile can include the types of allergens released from the matrix and / or the relative amounts of allergens released.

[0112] In one embodiment, the methods described herein indicate the release of allergens from the surface of a matrix (e.g., nanoparticle) by detecting a burst of peptides in the dissolution or release medium near the start of the assay. If the allergen is present within the matrix, the detection of the allergen will be more gradual over time based on the release rate. Allergens that are both present on the surface of the matrix and encapsulated within the matrix can be identified by an initial burst of peptide detection in the dissolution or release medium near the start of the assay as antigens from the surface are released from the matrix, followed by a more gradual increase in peptide detection in the dissolution or release medium as the allergen is released from the interior of the matrix (e.g., leaked).

[0113] As used herein, "dissolution medium" and "release medium" refer to compositions used to provide in vitro drug release information. Dissolution or release media can be used, for example, in quality control testing of samples to determine the release and / or stability of allergens in the sample. When selecting an appropriate dissolution or release medium, it is useful to determine the analytical target profile of the allergen (e.g., delayed release, constant release, extended release, etc.) and / or the allergen solubility profile. For a review of dissolution medium selection, see Martin and Gray (Summer 2011) Journal of Validation Technology.

[0114] As used herein, "release rate" refers to the rate at which the encapsulated peanut allergen material flows out of the composition and into the surrounding medium during an in vitro release assay. In one exemplary embodiment, the composition is first prepared for release testing by placing it in a suitable in vitro release medium. This is typically performed by exchanging the buffer after centrifugation to precipitate the matrix (e.g., synthetic nanocarriers) and reconstituting the matrix using mild conditions. In certain embodiments, the assay is initiated by placing the sample in an appropriately temperature-controlled apparatus at 37°C. Samples are typically removed at various time points.

[0115] As used herein, "release profile" refers to the types of proteins and / or peptides, e.g., peanut allergens, released from a matrix or container over time, and may also include the rate at which each specific protein, peptide, and / or allergen in or on the matrix or container is released.

[0116] "Exhibits pH-sensitive dissociation" means that the coupling between two entities (e.g., a peanut allergen and a matrix, such as a carrier molecule) is significantly reduced or eliminated as a result of exposing the two entities to a change in ambient pH. In certain embodiments, the relevant pH-sensitive dissociation can satisfy any relationship provided herein or a combination thereof.

[0117] In certain exemplary embodiments, the pharmaceutical composition comprises nanocarriers and / or microcarriers, e.g., synthetic nanocarriers and / or synthetic microcarriers. As used herein, "synthetic nanocarriers" or "synthetic microcarriers" refer to discrete objects having at least one dimension less than or equal to 5 microns.

[0118] In some embodiments, mass balance is compared between one or more peanut allergens in one or more compositions to be compared, e.g., in samples taken from the compositions over a period of time spanning different time points, or between different batches prepared at different times and / or by different methods. Comparative data can be expressed as relative quantification, and is typically expressed as a fraction or percentage.

[0119] Pharmaceutical compositions containing peanut allergens can generally be formulated with carriers, excipients, and other agents that provide suitable transfer, delivery, tolerance, etc. Exemplary formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicles containing lipids (cationic or anionic) (e.g., LIPOFECTIN™), anhydrous absorption creams, oil-in-water and water-in-oil emulsions, emulsions of carbowax (polyethylene glycol of various molecular weights), semisolid gels, and semisolid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J. Pharm Sci. Technol. 52: 238-311.

[0120] A "pharmaceutically acceptable excipient" refers to a pharmacologically inactive substance added to a composition (e.g., a therapeutic composition) to further facilitate administration of the composition. Pharmaceutically acceptable excipients include, but are not limited to, calcium carbonate, calcium phosphate, various diluents, various sugars and starch types, cellulose derivatives, gelatin, vegetable oils, polyethylene glycol, and the like.

[0121] "Dosage form" refers to a drug (eg, one or more peanut allergens) in a medium, carrier, vehicle, or device suitable for administration to a subject.

[0122] An "effective amount" refers to an amount effective for a purpose. For example, when an effective amount is used for therapeutic purposes, the effective amount is an amount that treats, alleviates, ameliorates, relieves, delays the onset of, inhibits the progression of, reduces the severity of, and / or reduces the incidence of one or more symptoms or features of a disease, disorder, and / or condition provided herein, such as peanut allergy.

[0123] "Subject" refers to animals, including mammals such as humans and non-human primates; birds; domesticated livestock or farm animals such as cats, dogs, sheep, goats, cows, horses, and pigs; laboratory animals such as mice, rats, and guinea pigs; fish; and the like.

[0124] As used herein, "vaccine" refers to a composition of matter that improves the immune response to a specific disease or condition. Vaccines typically contain factors that stimulate the subject's immune system to recognize a specific antigen (e.g., a peanut antigen) as foreign and eliminate it from the subject's body. Vaccines also establish immune "memory" so that if the subject is challenged again, the antigen will be quickly recognized and responded to. Vaccines can be prophylactic (e.g., to prevent future infection with any pathogen) or therapeutic (e.g., a vaccine against a peanut antigen is used to treat peanut allergies).

[0125] "Administering" or "administering" means providing a drug to a subject in a pharmacologically useful manner.

[0126] As used herein, "epitope" refers to a binding site comprising an amino acid sequence motif of about 6 to 15 amino acids that can be bound by an immunoglobulin (e.g., IgE, IgG, etc.) or recognized by a T-cell receptor when presented by an APC in conjunction with the major histocompatibility complex (MHC). A linear epitope is an epitope in which amino acids are recognized in the context of a simple linear sequence. A conformational epitope is an epitope in which amino acids are recognized in the context of a specific three-dimensional structure. Peanut allergens characterized by the methods of the present invention may comprise one or more peanut epitopes. Immunogenic epitopes can elicit an immune response, such as an allergic reaction, in vivo.

[0127] As used herein, an "immunodominant epitope" refers to an epitope bound by antibodies in a large proportion of the sensitized population, or an epitope where antibody titers are high, as a percentage or titer relative to antibody reactivity to other epitopes present in the same antigen. In one embodiment, the immunodominant epitope is bound by antibodies in greater than 50% of the sensitized population, more preferably greater than 60%, 70%, 80%, 90%, 95%, or 99% of the sensitized population. Peanut allergen signatures identified by the characterization methods of the present invention typically comprise one or more peanut immunodominant epitopes.

[0128] It will be apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. Having now described certain embodiments in detail, these embodiments will be more clearly understood by reference to the following examples, which are included herein for purposes of illustration only and are not intended to be limiting.

[0129] Example 1

[0130] Peanut Allergen Characteristics

[0131] The following describes the development and initial preliminary validation of a method for the relative quantification of four peanut allergens, Ara h1, Ara h2, Ara h3, and Ara h6. The method utilizes liquid chromatography coupled to tandem mass spectrometry and is based on the measurement of representative tryptic peptides from each of these proteins. In each case, the peptides were selected to encompass the majority of reported allergen protein isoforms.

[0132] The analytical method depends on the digestion reproducibility and digestion efficiency of the target protein in a complex protein mixture. Not all proteolytic peptides are good candidates for quantification, and good ionization efficiency and consistent trypsin cleavage and a low probability of peptides with post-translational modifications are important aspects of the selection process. Problems such as interference from other peptides in complex digests and other matrix effects are more difficult to predict. Once selected, a series of heavy isotope-labeled peptides and non-labeled synthetic forms of the selected peptides are obtained and used as internal standards and buffer calibration curves, respectively.

[0133] Peanut extracts prepared from lightly roasted peanut flour (Golden Peanut and Tree Nuts, Alpharetta, GA) were digested with trypsin in quadruplicate, and the standard was added to the digested samples. The standard contained two Ara h1 peptides, two Ara h2 peptides, two Ara h3 peptides, and two Ara h6 peptides. The peptides in the internal standard were 13 C and 15 One or both labels in N. The sequences of the internal standards are provided in Tables 1 and 2 below.

[0134] Table 1. Unlabeled synthetic "light" peptides used as internal standards

[0135] Peptide sequence peptides Chemical formula DLAFPGSGEQVEK ARA_L1-1 C60 H93 N15 O22 GTGNLELVAVR ARA_L1-2 C48 H85 N15 O16 GAGSSQHQER ARA_L2-1 C40 H65 N17 O17 QQEQQFK ARA_L2-2 C40 H62 N12 O14 RPFYSNAPQEIFIQQGR ARA_L3-1 C93 H139 N27 O26 AHVQVVDSNGNR ARA_L3-2 C52 H86 N20 O19 IMGEQEQYDSYDIR ARA_L6-1 C74 H111 N19 O28 S1 QMVQQFK ARA_L6-2 C40 H65 N11 O11 S1

[0136] Table 2. Labeled synthetic "heavy" peptides used as internal standards

[0137] Peptide sequence peptides Chemical formula DLAFPGSGEQVEK* ARA_H1-1 13C6 C54 H93 15N2 N13 O22 GTGNLELVAVR* ARA_H1-2 13C6 C42 H85 15N4 N11 O16 GAGSSQHQER* ARA_H2-1 13C6 C34 H65 15N4 N13 O17 QQEQQFK* ARA_H2-2 13C6 C34 H62 15N2 N10 O14 RPFYSNAPQEIFIQQGR* ARA_H3-1 13C6 C87 H139 15N4 N23 O26 AHVQVVDSNGNR* ARA_H3-2 13C6 C46 H86 15N4 N16 O19 IMGEQEQYDSYDIR* ARA_H6-1 13C6 C68 H111 15N4 N14 O28 S1 QMVQQFK* ARA_H6-2 13C6 C34 H65 15N4 N7 O11 S1

[0138] K* and R* are heavy isotope-labeled amino acids in which carbon-12 has been replaced by carbon-13 and nitrogen-14 by nitrogen-15, yielding 13C6 15N2 for lysine and 13C6 15N4 for arginine.

[0139] It is worth noting that alternative peptides can be selected, and specific characteristics of proteins make it difficult to achieve all desired features for all peptides. For example, both peptides used for Ara h6 contained methionine, which is undesirable but difficult to avoid because Ara h6 contains an unusually high content of sulfur-containing amino acids. Similarly, Ara h3 contains a stable N-terminal "missed" cleavage site that has been discovered. Typically, trypsin cleavage sites in close proximity to each other will result in preferential cleavage at one site, rather than both.

[0140] An internal standard was used to quantify the digest products. A buffer calibration curve of a synthetic, unlabeled signature peptide was generated, where a heavy isotope-labeled internal standard was added to the peanut extract digest and the calibration curve. Relative quantification was performed using the internal standard and the calibration curve to determine the relative amount of the signature peptide in the peanut extract digest.

[0141] The results were analyzed by nanoHPLC-MS-MS gas phase fragmentation (Orbitrap ELITE TM , ThermoScientific) were used to analyze samples containing digested test allergens and standards. Using the top-15 method, one sample (out of a total of four samples) was analyzed three times, and the remaining three samples were analyzed once to generate a total of six data files. TM These data files were analyzed using the ELISA software (Thermo Scientific). Application of the top-15 approach to identify the most abundant proteins ensured that peptides with high ionization and fragmentation efficiencies were identified in a consistent manner across the dataset.

[0142] The identities of the resulting fragments were determined using the UniProt sequence database at uniprot.org.

[0143] More than 90% of all known peanut allergens were detected in peanut flour samples, including Ara h1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, Ara h8, Ara h10, Ara h11, Ara h14, Ara h15, and Ara h lectin. The survey of identified allergens is described in Tables 3-18 below.

[0144] Ara h1, Ara h2, Ara h3, and Ara h6 represent the major peanut allergens.

[0145] Table 3. Ara h1 isoforms detected in peanut flour

[0146] Peanut allergens UniProt Ref Peaks identified by nanoHPLC-MS-MS Ara h 1 B3IXL2 no Ara h 1 E5G076 yes Ara h 1 N1NEW2 no Ara h 1 N1NG13 no Ara h 1 P43237 yes Ara h 1 Q6PSU3 yes Ara h 1 Q6PSU4 yes Ara h 1 Q6PSU5 yes Ara h 1 Q6PSU6 yes Ara h 1.0101 P43238 yes

[0147] Table 4. Ara h2 isoforms detected in peanut flour

[0148] Peanut allergens UniProt Ref Peaks identified by nanoHPLC-MS-MS Ara h 2 C0LJJ1 no Ara h 2.0101 Q6PSU2 yes Ara h 2.0201 Q6PSU2 yes

[0149] Table 5. Ara h3 isoforms detected in peanut flour

[0150]

[0151]

[0152] Table 6. Ara h5 isoforms detected in peanut flour

[0153] Peanut allergens UniProt Ref Peaks identified by nanoHPLC-MS-MS Ara h 5 D3K177 yes Ara h 5 L7QH52 yes Ara h 5 Q5XXQ5 yes Ara h 5.0101 Q9SQI9 yes

[0154] Table 7. Ara h6 isoforms detected in peanut flour

[0155] Peanut allergens UniProt Ref Peaks identified by nanoHPLC-MS-MS Ara h 6 A1DZE9 yes Ara h 6 A5Z1R0 no Ara h 6.0101 Q647G9 yes

[0156] Table 8. Ara h7 isoforms detected in peanut flour

[0157] Peanut allergens UniProt Ref Peaks identified by nanoHPLC-MS-MS Ara h 7 Q647G8 yes Ara h 7.0101 Q9SQH1 yes Ara h 7.0201 B4XID4 yes

[0158] Table 9. Ara h8 isoforms detected in peanut flour

[0159]

[0160]

[0161] Table 10. Ara h9 isoforms detected in peanut flour

[0162] Peanut allergens UniProt Ref Peaks identified by nanoHPLC-MS-MS Ara h 9.0101 B6CEX8 yes Ara h 9.0201 B6CG41 yes

[0163] Table 11. Ara h10 isoforms detected in peanut flour

[0164] Peanut allergens UniProt Ref Peaks identified by nanoHPLC-MS-MS Ara h 10.0101 Q647G5 yes Ara h 10.0102 Q647G4 yes

[0165] Table 12. Ara h11 isoforms detected in peanut flour

[0166] Peanut allergens UniProt Ref Peaks identified by nanoHPLC-MS-MS Ara h 11.0101 Q45W87 yes Ara h 11.0102 Q45W86 yes

[0167] Table 13. Ara h12 isoforms detected in peanut flour

[0168] Peanut allergens UniProt Ref Peaks identified by nanoHPLC-MS-MS Ara h 12.0101 EY396089 no

[0169] Table 14. Ara h13 isoforms detected in peanut flour

[0170] Peanut allergens UniProt Ref Peaks identified by nanoHPLC-MS-MS Ara h 13.0101 EY396019 no

[0171] Table 15. Ara h14 isoforms detected in peanut flour

[0172] Peanut allergens UniProt Ref Peaks identified by nanoHPLC-MS-MS Ara h 14.0101 Q9AXI1 yes Ara h 14.0102 Q9AXI0 yes Ara h 14.0103 Q6J1J8 yes

[0173] Table 16. Ara h15 isoforms detected in peanut flour

[0174]

[0175]

[0176] Table 17. Ara h lectin isoforms identified in peanut flour

[0177] Peanut allergens UniProt Ref Peaks identified by nanoHPLC-MS-MS Ara h lectin P02872 yes Ara h lectin Q38711 yes Ara h lectin Q43373 yes Ara h lectin Q43375 yes Ara h lectin Q8W0P8 yes

[0178] Table 18. Ara h hypothetical isoforms identified in peanut flour

[0179] Presumed peanut allergen UniProt Ref Peaks identified by nanoHPLC-MS-MS Ara i 2 A5Z1Q9 no Ara i 6 A5Z1Q6 no Ara d 2 A5Z1Q8 no Ara d 2 A8VT41 no Ara d 2 A8VT44 no Ara d 2 A8VT45 no Ara d 2 A8VT50 no Ara d 6 A5Z1Q5 no

[0180] Peptides of the major allergens are identified to form a collection of peptides that are used as indicators of the peanut allergen content in the composition (e.g., a "peanut peptide signature"). An ideal peptide signature uses peptides that represent fully cleaved true trypsin digests (with the possible exception of peptides containing consecutive arginine and / or lysine residues), with one or the other cleavage site generally being preferentially cleaved. The selected peptides also generally have sequence conservation with a false discovery rate (FDR) of less than 1% and sequence conservation across multiple isoforms. In addition, the selected peptides generally have no or minimal post-translational modifications, including oxidation and glycosylation, and are of high quality for manual curation based on tandem mass spectrometry (MS-MS). Isoforms are represented by genetic variation, including peptides resulting from truncated or deleted sequences. Examples of high quality peptides identified by MS-MS are Figures 1A to 1C The Ara h 1 peptide shown in .

[0181] For example, Ara h1k heterogeneity was determined by comparing sequence variation within UniProt database records. Exemplary Ara h1 allergen digests identified by UniProt reference number P43238 are shown in Table 19. Sequences from other UniProt Ara h1 records were also compared (see Table 20). Preferred Ara h1 allergen digests were selected based on their usefulness in peanut allergen characterization based on: 1) a preference for zero missed trypsin cleavage sites; 2) presence in all sequences with a false discovery rate (FDR) of less than 1%; 3) absence or minimal post-translational modification sites; 4) high-quality fragments as determined by manual analysis of tandem mass spectrometry (MS-MS) data; 5) indication that the sequence is unique within the peanut proteome by BLAST searches; and 6) the sequence has the greatest number of isoforms. The resulting initial list of allergen digests was identified as the most useful candidates for analysis of peanut antigens in peanut flour extracts, as shown in Tables 20-23, representing peanut allergens from Ara h1, Ara h2, Ara h3, and Ara h6, respectively.

[0182] Table 19. Ara h1 allergen digestion products from UniProt Ref. P43238

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189] Table 20. Ara h1 sequences identified in prepared peanut extracts

[0190]

[0191]

[0192] Table 21. Ara h2 sequences identified in prepared peanut extracts

[0193]

[0194] Table 22. Ara h3 sequences identified in prepared peanut extracts

[0195]

[0196]

[0197]

[0198] Table 23. Ara h6 sequences identified in prepared peanut extracts

[0199]

[0200] Equivalent content

[0201] The present disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics of the present disclosure. The foregoing embodiments are therefore to be considered in all respects as illustrative rather than restrictive of the present disclosure. The scope of the present disclosure is therefore indicated by the appended claims rather than by the foregoing description, and all variations that come within the meaning and range of equivalents of the claims are intended to be embraced herein.

Claims

1. A method for characterizing peanut allergens in an aqueous medium, comprising: digesting peanut allergens present in an aqueous medium to produce allergen digestion products; fragmenting allergen digests to produce peptide fragments; and determining a profile of allergenic digestion products of a peanut allergen in said aqueous medium by detecting said peptide fragments, wherein said profile comprises each of Ara h 1, Ara h2, and Ara h6 digestion products, wherein the signature comprises allergen digests from each of Ara h 1, Ara h2, and Ara h6, having polypeptides as set forth in SEQ ID NO: 17, SEQ ID NO: 70, and / or SEQ ID NO: 177 from Ara h 1; SEQ ID NO: 197 and / or SEQ ID NO: 198 from Ara h 2; and SEQ ID NO: 236 and / or SEQ ID NO: 237 from Ara h 6, and wherein the peanut allergen is digested using trypsin.

2. The method according to claim 1, wherein the aqueous medium is a dissolution medium, a release medium or an analytical sample, and / or wherein the aqueous medium further comprises an internal standard.

3. The method of claim 2, wherein the internal standard comprises one or more heavy isotopes.

4. The method according to claim 1, further comprising the step of comparing the signature with a signature standard, and / or wherein fragmenting the allergen digestion product and determining the signature is performed by a method selected from the group consisting of one or any combination of LC-MS, LC-MS-MS, nano-LC-MS-MS and nanoHPLC-MS-MS.

5. The method of claim 1, wherein the allergen digestion product is 4 to 50 amino acids in length.

6. The method of claim 1, wherein the allergen digestion product is 6 to 30 amino acids in length.

7. The method of claim 1, wherein the allergen digestion product is 6 to 15 amino acids in length.

8. The method of claim 1, wherein the signature comprises a digestion product that is free of missed proteolytic cleavage sites.

9. The method of claim 1, wherein the allergen digestion products are present in isoforms of Ara h1, Ara h2 and Arah6.

10. A method for determining the in vitro release profile of a peanut allergen from a matrix into an aqueous medium, comprising: obtaining a sample from the aqueous medium at each of a plurality of time points; digesting peanut allergens present in the sample to produce an allergen digestion product; fragmenting allergen digests to produce peptide fragments; and detecting the peptide fragments at at least two of the plurality of time points to identify the allergen digestion products, thereby determining an in vitro release profile of the peanut allergen into the aqueous medium, wherein the release profile comprises each of Ara h 1, Ara h 2, and Ara h 6 digestion products, wherein the in vitro release profile comprises allergen digestion products from each of Ara h 1, Ara h 2 and Ara h 6 having a polypeptide as shown in SEQ ID NO: 17, SEQ ID NO: 70 and / or SEQ ID NO: 177 from Ara h 1; SEQ ID NO: 197 and / or SEQ ID NO: 198 from Ara h 2; SEQ ID NO: 236 and / or SEQ ID NO: 237 from Ara h 6, and wherein the peanut allergen is digested using trypsin.

11. The method of claim 10, wherein the aqueous medium is a dissolution medium, a release medium, or an analytical sample.

12. The method of claim 10, wherein the allergen digestion product is 4 to 50 amino acids in length.

13. The method according to claim 10, wherein fragmenting the allergen digestion products and detecting the peptide fragments are performed by a method selected from the group consisting of liquid chromatography-tandem mass spectrometry (LC-MS-MS), nanoLC-MS-MS and nanohigh performance liquid chromatography-tandem mass spectrometry (nanoHPLC-MS-MS) or a combination thereof.

14. The method of claim 10, wherein the aqueous medium further comprises an internal standard, wherein the internal standard optionally comprises one or more heavy isotopes.

15. The method of claim 10, wherein the matrix comprises one or both of nanoparticles and microparticles.

16. The method of claim 10, wherein the release profile is obtained over a period of three hours.

17. The method of claim 10, wherein the allergen digestion product is 6 to 30 amino acids in length.

18. The method of claim 10, wherein the allergen digestion product is 6 to 15 amino acids in length.

19. The method of claim 10, wherein the in vitro release profile comprises allergen digestion products without omitted proteolytic cleavage sites.

20. The method of claim 10, wherein the allergen digestion products are present in isoforms of Ara h 1, Ara h2, and Ara h6.

21. The method of claim 10, wherein the release profile is obtained over a period of six hours.

22. The method of claim 10, wherein the release profile is obtained over a period of twelve hours.

23. The method of claim 10, wherein the release profile is obtained over a period of twenty-four hours.

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