HK1227761A1 - A method for the production of hydrolyzed allergen - Google Patents

Abstract

The present invention discloses a method for the production of hydrolyzed allergen. Specifically, a method for the production of a purified extract of natural allergens comprising the steps of: extracting a natural source of allergens comprising allergenic proteins to form an extract; purifying of said extract to remove non-protein components to form a purified extract; denaturating said purified extract to form a purified denaturated extract, said purified denaturated extract comprising proteins, wherein the most abundant proteins, forming together at least 60% of all proteins, are at least two proteins, and all proteins represent at least 60% of the dry weight of the purified denaturated extract and a method for the production of a purified extract of natural allergens comprising the steps of: hydrolysing a denaturated allergen to form an allergen hydrolysate; purifying said allergen hydrolysate to remove peptides with a molecular weight above 10,000 Da and below 1,000 Da in order to obtain a purified hydrolysate where 70%, more preferably 80% of the peptides are between 10,000 Da and 1,000 Da.

Description

Method for preparing hydrolyzed allergen

The application is a divisional application of Chinese patent application with the application number of 200780023913.3, the application date of 2007, 6 and 28 months, and the title of the application is 'method for preparing hydrolyzed allergen'.

Technical Field

The present invention relates to a method for producing an extract of natural allergen, peptides derived from these extracts, and an allergen extract obtainable by a novel method.

Background

Common allergens are pollen, dust mites, molds, pharmaceuticals, food and animal hair and dander.

The most common allergic diseases are rhinitis, asthma and atopic dermatitis. Allergic asthma is a chronic inflammatory disorder. Symptomatic treatment of allergic disorders is achieved through the use of antihistamines, beta-antagonists and corticosteroids.

Furthermore, so-called "specific" immunotherapy is based on desensitization. Subcutaneous injections of specific offensive (inflicting) allergens are typically administered to patients. Treatment was initiated with a small allergen dose and the dose was increased. Treatment is usually maintained for several years. Such treatments suffer from pure patient compliance and are questioned for safety reasons because of the severe allergic reactions experienced by patients.

In addition to methods involving repeated subcutaneous injections, there are methods of oral desensitization.

U.S. Pat. No. 4,822,611 discloses a method of treating allergy involving oral treatment with an allergen. U.S. Pat. No. 4,822,611 describes a method for displaying batch-to-batch variation of extracts from different manufacturers and different commercially available "bulk" allergenic extracts. The preparation of these extracts is not described.

GB 1247614 discloses a process for extracting allergens. The aim of the method is to obtain a more complete and effective allergenic extract by including all extractable allergenic components.

U.S. Pat. No. 5,770,698 discloses a method for purifying an extract of allergenic active proteins. The spectrum of FIG. 2 is not shown 280_nmPeak of (2). This means that the extract contains significant levels of non-protein impurities. WO 99/22762 discloses a similar process, whereby the product also contains a large amount of non-protein impurities.

On the other hand, there is a trend towards the development of highly specific formulations based on single epitopes. For example, WO00/58349 discloses isolated and purified peptides comprising two peptide bonds remote from the leucine of a tyrosine/arginine pair. These peptides can be used to prepare pharmaceutical compositions to achieve treatment or prevention, in this case especially for dog allergy in dogs.

In one aspect, methods are used to purify a particular recognized single sensitizing molecule. On the other hand, attempts have been made to produce allergenic extracts which are as complete as possible.

According to the first option, it has always been possible that an allergen preparation has no relevant epitope to induce tolerance in a defined patient. The second option has the disadvantage of lot-to-lot variation and the disadvantage of the presence of compounds capable of eliciting an immune response (such as DNA molecules, carbohydrates, lipids of their complexes).

Disclosure of Invention

It is an object of the present invention to overcome at least some of the disadvantages of the prior art, in particular to provide antigens from natural allergens which have a significantly reduced capacity to elicit an allergenic response compared to a crude allergen extract, but which are also capable of stimulating T-cells.

The problem is solved by providing a method for the preparation of an allergen extract comprising most proteins (which proteins comprise part of the allergen extract) but having a reduced, preferably very low content of non-protein components (such as nucleic acids, lipids, sugars, etc.).

The extracts prepared according to the invention are superior to prior art extracts, in particular they show a reproducible protein composition without being purified to a single epitope.

The preparation method of the allergen extract comprises the following steps:

a) extracting a natural source of allergen comprising an allergenic protein to form an extract,

b) purifying the extract solution to remove non-protein components to form a purified extract solution,

c) denaturing the purified extract to form a purified denatured extract,

the purified denatured extract comprises proteins, wherein the most abundant (w/w) proteins (which together form at least 60% (w/w) of all proteins) are at least two proteins, and all proteins make up at least 60% (w/w) of the purified denatured extract's dry weight.

This method is called method I.

In contrast to prior art methods, the method of the present invention produces an allergen extract comprising mainly proteins without purifying the extract to mono-peptides or proteins.

In contrast to the products of the prior art, the products of the invention have the following advantages:

-substantially removing immunological material other than protein;

natural allergen extracts are capable of stimulating T-cells with a reduced capacity to elicit rapid allergic reactions (basophil activation, mast cell degranulation).

Different natural allergens can be used as starting materials. Typical natural starting materials are milk, venom, eggs, weeds, grasses, trees, shrubs, flowers, vegetables, grains, fungi, fruits, berries, nuts, seeds, beans, fish, shellfish, seafood, meat, spices, insects, mites, molds, animals, pigeon ticks, worms, cockscomb, animal dander, nematodes, hevea brasiliensis, and mixtures thereof.

After material extraction, the extract is purified to remove non-protein components such as sugars, lipids, nucleic acids, and the like. Typically several different proteins are present as protein fractions of the purified extract.

According to the prior art, one protein is purified and the other remaining proteins are "impurities".

Rather, the object of the present invention is to purify proteins together. The relative content of proteins in the purified extract can be easily measured using methods such as SDS-PAGE followed by densitometry.

For 60% of the total weight of proteins, it is desirable to bind at least the two most predominant proteins, i.e., no single protein comprises 60% (w/w) or more of all proteins. More preferably, 60% of all proteins are formed by at least 3 major proteins, preferably by at least 4 major proteins and more preferably by at least 5, 6, 7, 8, 9 or 10 proteins.

For example, the following proteins:

protein 1: 27 percent of

Protein 2: 13 percent of

Protein 3: 34 percent of

Protein 4: 19 percent of

Protein 5: 17 percent of

The most predominant protein that together forms at least 60% (≈ 60% or more) is protein 3+1(34+27 ═ 61%).

Furthermore, the total protein content of the purified extract is at least 60 wt.% of the purified extract, preferably at least 70 wt.% or 80 wt.%, more preferably 90 wt.% of the purified extract.

Preferably, the extraction is carried out with an aqueous solution. Suitable salts are, but not limited to, for example, carbonates, bicarbonates, phosphates, acetates, TRIS and HEPES.

Also unlike many other extraction methods, it is preferred that the amount of extraction medium is relatively large, i.e. at least 20 times the weight of the natural source of the allergen, preferably 100 times or more said weight.

Purification of the extract may be carried out by one or more of:

ion exchange chromatography steps (including anion exchange chromatography and cation exchange chromatography),

a size exclusion chromatography step (also known as gel filtration),

-a precipitation step of the precipitate,

-a hydrophobic interaction chromatography step,

pseudo-affinity and affinity chromatography, and/or

-diafiltration.

In a preferred embodiment ion exchange chromatography is used, wherein the pH of the loading solution in the case of a cation exchanger is between the acidic functional pKa of the cation exchanger and the pKa of the protein having the lowest pKa of the proteins in the extraction solution. In the case of an anion exchanger, the pH lies between the basic functional pKa of the anion exchanger and the pKa of the protein with the highest pKa of the proteins that make up the extract.

By this method, all proteins are bound to the ion exchanger, and neutral impurities as well as impurities having the same charge as the ion exchange resin are removed.

In a preferred embodiment, at least one purification step is performed with a solution comprising one or more surfactants and/or denaturants. The surfactant may be nonionic, anionic, cationic or amphoteric. Suitable denaturants are chaotropic agents, reducing agents and mixtures thereof. Suitable denaturants are, for example, urea, guanidine hydrochloride, ethylene glycol, isopropanol. Suitable concentrations of urea are 3M or higher, preferably 4M or higher. Suitable concentrations of guanidinium salts are preferably 2M, preferably 3M or higher. Suitable concentrations of ethylene glycol and/or isopropanol are 5 wt% or higher, more preferably 10 wt% or higher, up to 20 wt%.

In some cases, it is sufficient to prepare a purified extract according to method I of the invention. The extract can be used for in vitro/in vivo and in vitro diagnosis, and for preventing and treating allergic diseases. A further embodiment of the invention is a process for the preparation of an allergen hydrolysate from the extract of Process I or from any other source. If the extract is from any other purified allergen source than from method I, a preliminary step of denaturation is required to improve digestibility.

The process (process II) comprises the following steps

a) Hydrolyzing the denatured allergen to form an allergen hydrolysate,

b) purifying the allergen hydrolysate to remove peptides having a molecular weight above 10,000Da and below 1,000Da, thereby obtaining a purified hydrolysate wherein 70%, more preferably 80% of the peptides are between 10,000Da and 1,000 Da.

The advantage of the product thus obtained is that the peptide is the result of digestion of denatured proteins. Due to the specific size scaling, the peptides have a reduced capacity to induce rapid allergic reactions as well as pro-inflammatory reactions.

If desired, the denaturation is preferably carried out in the presence of a chaotropic agent, a reducing agent or a mixture thereof. Suitable chaotropic agents are, for example, urea and guanidine hydrochloride. Typical reducing agents are, for example, dithiothreitol (dithiotriethol), beta-mercaptoethanol, thioglycerol, and mixtures thereof.

The hydrolysis step is usually carried out with enzymes. Suitable enzymes are, for example, pepsin, insulin, chymotrypsin. The hydrolysis step can also be carried out in the presence of a chaotropic agent, preferably urea or guanidine hydrochloride. The concentration of urea and guanidine hydrochloride during hydrolysis should be less than 4M, preferably less than 3M.

In step b) of method II, peptides with a molecular weight greater than 10,000Da or less than 1,000Da are removed.

Thus, the peptides of the purified hydrolysate comprise peptides having a molecular weight between 1,000 and 10,000 Da. Suitable methods for removing large or small peptides are ultrafiltration and size exclusion chromatography. The size exclusion chromatography may also be performed in the presence of chaotropic agents such as urea, guanidine hydrochloride, ethylene glycol, isopropanol and mixtures thereof.

A further embodiment of the present invention is an allergen extract obtainable by method I of the present invention. Typically in this extract the most predominant proteins by weight (which together form at least 60% by weight of all proteins) are at least 2 proteins, preferably at least 3 or 4 proteins or more preferably at least 5, 6, 7, 8, 9 or 10 proteins. Pass optical density 260_nmPurity was seen: optical Density 280_nm-ratio of<1, preferably<0.9, more preferably between 0.75 and 0.9.

A further embodiment is the allergen hydrolysate obtainable by process II. The allergen hydrolysate can be used for:

-in vivo diagnosis of allergic diseases: prick test, intradermal injection, conjunctival, inspiratory and inhalational test;

in vitro and in vitro diagnosis of allergic diseases: ELISA kits or standard samples used in the assay;

-prevention and treatment of allergic diseases: for desensitization/desensitization therapy and modulation of immune responses with/without adjuvant.

The allergen extract of the present invention can be used in the preparation of pharmaceutical compositions and/or food compositions to induce tolerance. Induction of tolerance can be used to treat and prevent allergic reactions.

A further embodiment of the invention is a pharmaceutical composition comprising an allergen extract of the invention in intact or hydrolysed form. In addition, the pharmaceutical composition may comprise one or more of the following: nucleoside triphosphates, nucleoside diphosphates, nucleoside monophosphates, nucleic acids, peptide nucleic acids, nucleosides or their analogs, immunosuppressive cytokines, compounds that induce expression of immunoproteasome, 1, 25-dihydroxyvitamin D3 or their analogs, lipopolysaccharide, endotoxin, heat shock protein, thioredoxin with NADPH or NADP-thioredoxin reductase, dithiothreitol, adrenergic receptor such as albuterol, adrenergic receptor such as butoxylamine, adhesion-regulating molecule ICAM-1, N-acetyl-L-cysteine, y-L-glutamyl-L-cysteinyl-glycine (reduced L-glutathione), compounds of expression of alpha-2-macroglobulin, inducers of expression of Foxp3 gene, flavonoids, Isoflavones, pterocarpans (pterocarpans), stilbenes such as resveratrol, tachykinin receptor antagonists, chymase inhibitors, vaccine adjuvants like CpG or MPL or tolerogenic adjuvants like zymosan, beta-1, 3-glucan, regulatory T-cell inducers, muco-adhesion agents (such as phytolectins, zinc salts, polysaccharides, vitamins and bacterial lysates) that bind particles to the intestinal mucosa.

Based on the natural allergen source in the composition, allergens selected from pollen allergens, cow milk allergens, venom allergens, egg allergens, weed allergens, grass allergens, tree allergens, shrub allergens, flower allergens, vegetable allergens, grain allergens, fungus allergens, fruit allergens, berry allergens, nut allergens, seed allergens, bean allergens, fish allergens, shellfish allergens, marine allergens, meat allergens, spice allergens, insect allergens, mite allergens, mold allergens, animal allergens, pigeon ticks allergens, worm allergens, cockscomb allergens, animal dander allergens, nematode allergens, hevea brasiliensis allergens may be included.

In a preferred embodiment, the pharmaceutical composition is prepared for oral administration, for sublingual drug delivery, for enteral drug delivery.

Drawings

FIG. 1: immunoreactivity as determined by IgG immunoblotting. Belt 1: molecular weight markers, band 2: crude protein extract, band 3: purified allergen denaturing extract. Membranes were blocked with 5% BSA and 3% cow's milk. Patient serum was diluted to 1/250. IgG binding was detected by goat anti-human IgG HRP diluted to 1/2,500 and revealed by TMB carrier. Allergen 1: 61-54kDa, allergen 2: plus or minus 36-31 kDa.

FIG. 2: immunoreactivity as determined by IgE immunoblotting. Belt 1: molecular weight markers, band 2: crude protein extract, band 3: a purified protein. Membranes were blocked with 5% BSA and 3% cow's milk. Patient serum was diluted to 1/5. IgE binding was detected by goat anti-human igekrp diluted to 1/10,000 and revealed by TMB vector. Allergen 1: 61-54kDa, allergen 2: plus or minus 36-31 kDa.

FIG. 3: SEC G25 elution profile exhibited a blocking peak. The ratio of column volume/sample volume was 12. The resin was equilibrated with tri-HCl 25mM, urea 1.5M, pH 8.0 at a flow rate of 9 ml/min. The elution was followed by absorbance at 280 nm.

FIG. 4: protein profile determined by SDS-PAGE. 4-12% bis-trisgelatin. Belt 1: molecular weight markers, band 2: purified allergen denaturing extract. Staining was performed using Coomassie Brilliant blue R-250.

FIG. 5: protein and peptide profiles determined by SDS-PAGE. 4-12% bis-trisgelatin. Belt 1: molecular weight markers, band 2: purified allergen denaturing extract (13 μ g), band 3: hydrolysate (13 μ g). Staining was performed using Coomassie Brilliant blue R-250.

FIG. 6: g50SEC elution profile. The column was equilibrated with urea 2M, NaCl 100mM, pH 3.0. The flow rate was 15 ml/min. The ratio of column volume/sample volume was 10. The elution was followed by absorbance at 280 nm.

FIG. 7: calibration curve for HPLC analysis. 10 microliters of the following standard samples (1 mg/ml) were injected onto a BioSep-SEC S2000 column: 1. bovine serum albumin (66kDa), 2. beta. -lactoglobulin (18.5kDa), 3. cytochrome C (12kDa), 4. glucagon (3.5kDa), 5.1kDa synthetic peptide.

FIG. 8: size exclusion HPLC curve. Column: BioSep-SEC S2000 (PhenomENEX). Elution buffer: na (Na)₂HPO₄50 mM-SDS 0.5% (w/v) pH 6.8. The flow rate was 1 ml/min. Detection was at 214 nm. 10 microliters of sample was injected. The percentage of the peptide of interest was calculated using the area under the curve between the 10kDa and 1kDa limits.

FIG. 9: allergenic properties of pollen-derived products. Blood samples from pollen allergic volunteers were incubated with increasing concentrations (0, 1, 10, 100 and 1000 ng/ml) of crude pollen extract, pollen purified protein and pollen purified peptide. Gp53 protein expression was determined by flow cytometry with gating on IgE-positive leukocytes. Results were expressed as percentage of gp53 positive cells in activated cells (mean ± deviation of 2 measurements).

FIG. 10: proliferative stimulation of human PBMCs by pollen proteins and pollen peptides. Incubation of pollen-allergic volunteers purified in the presence of increasing concentrations (1030 and 90. mu.g/ml) of pollen proteins or pollen peptides at 37 ℃Human PBMC for 5 days. Will 2³H]Thymidine was added to the cell culture medium for 16 hours, and the value was measured using the liquid scintillation principle using a β counter³H]Incorporation of thymidine. The results are expressed as the average of 5 measurements. The process of the present invention is further illustrated by the following non-limiting examples.

Detailed Description

Examples

Example 1: extraction of

1% (w/v) pollen (Lolium perenne from ALLERGON) was added to sodium bicarbonate (12.5mM) and incubated for 2 hours with stirring. The solution was then clarified by the addition of 2% (w/v) diatomaceous earth (ACROS) and filtered through a 0.2 micron filter. This sample constitutes the crude extract.

The extract was analyzed for the presence of allergens by immunoblotting using serum from pollen allergic patients. IgG and IgE epitopes were visualized with anti-human IgG or IgE antibodies.

As shown in fig. 1 and 2, there are two major allergens in the extract.

The crude extract was acidified to pH3.0 and Tween 20 (0.1%, v/v) was added. The sample constitutes the acidified extract.

Example 2: purification of allergen proteins

The allergen extract was purified by:

-cation exchange chromatography

Wells were equilibrated with 28 × bed volumes (Bv) of sodium bicarbonate 12.5mM, citrate 30mM, pH3.0, Tween 200.1% (v/v)Proton exchange membrane (sartobind) S^-The column was initially washed with 35 × Bv sodium bicarbonate 12.5mM, citrate 30mM, pH3.0, Tween 200.1% (v/v), then with 42 × Bv sodium bicarbonate 12.5mM, citrate 30mM, pH3.0, eluting the protein with carbonate 0.1M, sodium chloride 0.5M, pH 9.15, tracking the presence of protein by OD 280nm, concentrating the fractions of interest (FIG. 3).

Ammonium sulfate precipitation

This step is carried out at 0-4 ℃.

An amount of ammonium sulfate to 90% saturation was added to the product with stirring. Stirring was stopped after the salt had completely dissolved. The suspension was incubated overnight and centrifuged 2 times at 10,000g during 15 minutes. The supernatant was carefully discarded each time.

Denaturation

The microparticles were resuspended at 9 mg/ml in urea 6M, DTT 10mM, Tris HCl 0.1M, pH 8.0 and incubated at 37 ℃ for 1 hour.

Size exclusion chromatography on G25 resin (Fine Sephadex from AMERSHAM)

The denatured sample was loaded onto a column and the proteins were eluted with tri-HCl 25mM, urea 1.5M, pH 8.0.

The presence of protein was followed by OD measurement at 280 nm. The fractions of interest are pooled to form a purified denatured allergen extract.

The purified allergen extract was further analyzed. Protein content (BCA assay) and dry weight were determined to assess protein purity. By removal of carbohydrates (orcinol test) and also by OD₂₆₀/OD₂₈₀The reduction in purification efficiency is followed.

Table 1: removing non-protein components to form a purified extract

As shown in table 1, the purification process allowed:

-the protein percentage in the extract increases from-15% to 80%;

-OD₂₆₀/OD₂₈₀the ratio tends to 0.5 (which represents pure protein);

significant removal of carbohydrates (residual content represents the carbohydrate fraction of the protein).

FIG. 4 shows a typical SDS-PAGE profile obtained for purified denatured allergen extracts. As can be seen, 6 proteins accounted for at least 60% of the total weight of proteins in the purified extract.

Example 3: hydrolysis of denatured allergen extract

The extract was hydrolyzed using the following protocol:

acidifying the purified allergen extract to pH 2.0. The digestion was performed with 2.5 mg/ml pollen protein and 1eu. ph.u pepsin (MERCK) (for 337 mg protein) at 37 ℃ during 2 hours.

FIG. 5 shows a comparison between the purified extract (lane 2) and the hydrolysed extract (lane 3). As can be seen, after incubation with pepsin, the high molecular weight proteins corresponding to the denatured, undigested proteins disappeared.

Example 4: purification of

To remove peptides with MW ≥ 10,000Da and MW ≤ 1,000Da, the hydrolysate is purified by:

size exclusion chromatography on G50 resin (fine Sephadex from AMERSHAM).

16.5% (v/v) isopropanol and 0.1M NaCl were added to the hydrolysate. The sample was quickly loaded onto the G50 column. The peptides were eluted and fractions containing peptides (MW. ltoreq.10 kDa) were pooled as shown in FIG. 6.

Diafiltration on a 1kDa membrane (Ultrafiltration card Omega PES from PALL).

The peptide was concentrated 10 x, diafiltered with 10 volumes of tri-HCl 50mM pH 7.4, and finally concentrated 2.5 x. This sample constitutes a purified allergen hydrolysate.

The efficiency of purification was controlled by size exclusion HPLC. With Na₂HPO₄50 mM-SDS 0.5% (w/v) pH 6.8 BioSep-SEC S2000 column (PhenomENEX) was equilibrated at a flow rate of 1 ml/min. The peptide was detected at 214 nm.

The 10kDa and 1kDa limits were calculated from the calibration curves as illustrated in FIG. 7.

As shown in FIG. 8, peptides with molecular weights between 1,000Da and 10,000Da account for approximately 75% of all peptides in the purified hydrolysate.

Example 5: reduction of allergenicity

The allergenic properties of crude pollen extracts (according to example 1), purified pollen proteins (according to example 2) and purified pollen peptides (according to example 4) were assessed by measuring their ability to induce basophil degranulation.

Fresh human blood samples from pollen allergic volunteers incubated with increasing concentrations of crude pollen extract, purified protein and purified peptide were tested in vitro. Basophil degranulation was assessed by measuring, by flow cytometry, the expression of the gp53 protein marker on the cell membrane of activated cells (i.e., IgE positive cells). This protein is usually present in the membrane of the granule within the remaining cells and appears on the cell surface upon cell activation (due to fusion of the membrane of the granule with the cytoplasmic membrane). The protein thus becomes detectable by the labeled specific anti-gp 53 antibody. As shown in fig. 9, the purified peptides were about 30 x less allergenic than the purified proteins, and the purified peptides were 100 x less allergenic than the crude pollen extract.

Example 6: immunogenicity of pollen proteins and pollen peptides

The immunogenicity of allergen proteins and peptides was investigated by measuring their proliferation to stimulate human Peripheral Blood Mononuclear Cells (PBMCs).

PBMCs purified by density gradient centrifugation (blood samples from "pollen-sensitized" volunteers) were cultured for 5 days in 96-well plates in the presence of increasing concentrations of pollen proteins and pollen peptides. On day 5, the composition is administered³H]Thymidine was added to the cell culture medium and the plate was further incubated at 37 ℃ for 16 hours as determined using the liquid scintillation principle using a β counter³H]Incorporation of thymidine (FIG. 10).

Pollen proteins (according to example 2) and pollen peptides (according to example 4) stimulated proliferation of human PBMCs in a dose-concentration dependent manner. The proliferation induced by the allergen peptide is slightly lower than that observed in the response protein. These results show that the peptide preparation method maintains the epitopes of most allergens involved in T cell activation.

Claims (25)

1. A process for the preparation of a purified allergen hydrolysate, the process comprising the steps of:

-extracting a natural source of allergen comprising allergenic protein to form an extract,

-purifying the extract liquid to remove non-protein components to form a purified extract liquid,

-denaturing the purified extraction liquid to form a purified denatured extraction liquid, wherein the denaturation is performed using a mixture of a chaotropic agent and a reducing agent, the purified denatured extraction liquid comprising proteins, wherein the most abundant (w/w) proteins that together form 60% or more (w/w) of all proteins are at least two proteins, and all proteins account for at least 60% (w/w) of the purified denatured extraction liquid dry weight,

-hydrolyzing the purified denatured extract to form an allergen hydrolysate,

-purifying the allergen hydrolysate to remove peptides having a molecular weight above 10,000Da and below 1,000Da, thereby obtaining a purified hydrolysate, wherein 80% of the peptides are between 10,000Da and 1,000 Da.

2. The method of claim 1, wherein the extraction is performed in a solution that is salt free or comprises a salt selected from carbonate, bicarbonate, phosphate, acetate, TRIS, or HEPES.

3. The method according to claim 1 or 2, wherein the extraction is performed with an extraction medium, wherein the weight of the extraction medium is at least 20 times the weight of the natural source of the allergen.

4. The method according to claim 1 or 2, wherein the extraction is performed with an extraction medium, wherein the weight of the extraction medium is 100 times the weight of the natural source of the allergen.

5. The method of claim 1, wherein the purification comprises one or more ion exchange chromatography steps, gel filtration or size exclusion chromatography steps, precipitation steps, hydrophobic interaction chromatography steps, pseudo-affinity or affinity chromatography steps, or diafiltration steps.

6. The method of claim 5, wherein at least one purification step is performed using a solution comprising a surfactant and/or a denaturant.

7. The method of claim 6, wherein

The chaotropic agent is selected from the group consisting of: urea, guanidine hydrochloride, and mixtures thereof, and

wherein the reducing agent is selected from: dithiothreitol, thioglycerol, beta-mercaptoethanol, and mixtures thereof.

8. The method according to claim 7, wherein the concentration of urea is higher than 4M and/or the concentration of guanidine hydrochloride is more than 3M.

9. The method according to claim 7, wherein the concentration of urea is higher than 5M and/or the concentration of guanidine hydrochloride is more than 4M.

10. The method of claim 1 or 2, wherein the hydrolysis is performed using an enzyme.

11. The method of claim 1 or 10, wherein the hydrolysis is performed using pepsin, trypsin, or chymotrypsin.

12. A method according to claim 1 or 10, wherein hydrolysis is carried out in the presence of a chaotropic agent.

13. The method of claim 1 or 12, wherein the hydrolysis is performed in the presence of urea and guanidine hydrochloride.

14. The method according to claim 1 or 10, wherein the removal of peptides is performed by size exclusion chromatography and/or by ultrafiltration.

15. The method of claim 14, wherein the size exclusion chromatography step is performed in the presence of a chaotropic agent.

16. The method of claim 14, wherein the size exclusion chromatography step is performed in the presence of urea, guanidine hydrochloride, ethylene glycol, isopropanol, or a mixture thereof.

17. The method of claim 1 or 10, wherein

The allergen is selected from: pollen allergen, cow milk allergen, venom allergen, egg allergen, weed allergen, grass allergen, tree allergen, shrub allergen, flower allergen, vegetable allergen, grain allergen, fungus allergen, fruit allergen, berry allergen, nut allergen, seed allergen, bean allergen, fish allergen, shellfish allergen, marine allergen, meat allergen, spice allergen, insect allergen, mite allergen, mold allergen, animal allergen, pigeon tick allergen, worm allergen, sea cockscomb allergen, animal dander allergen, nematode allergen, and hevea brasiliensis allergen.

18. A purified allergen hydrolysate obtainable by the method of claim 1 or 10.

19. Use of the allergen hydrolysate according to claim 18 for the preparation of a pharmaceutical composition for inducing tolerance.

20. Use of the allergen hydrolysate of claim 18 in a food composition.

21. The use according to claim 19, wherein induction of tolerance is used to treat or prevent allergic reactions.

22. A pharmaceutical composition comprising the allergen hydrolysate of claim 18.

23. The pharmaceutical composition of claim 22, wherein the allergen is selected from the group consisting of pollen allergens.

24. The pharmaceutical composition according to claim 22, wherein the allergen is selected from the group consisting of mite allergens.

25. The pharmaceutical composition according to claim 22, for oral administration, for sublingual drug delivery, for enteral drug delivery.