Purification method of allergen extract

Abstract

The present invention relates to methods for producing semi-purified and purified allergen extracts, as well as pharmaceutical compositions and vaccines for use in the diagnosis and treatment of allergies. In one aspect of the present invention, a method for producing a decolorized allergen extract is provided. The method comprises: a) basifying a natural allergen extract; b) removing molecules with a molecular size of less than 3.5 kDa; and c) adjusting the pH to neutral to produce the decolorized allergen extract.

Description

[Technical field]

[0001] The present invention relates to methods for the preparation of semi-purified and purified allergen extracts as well as pharmaceutical compositions and vaccines for use in the diagnosis and treatment of allergies. [Background technology]

[0002] Allergy is an acquired hypersensitivity disorder of the immune system, triggered by exposure to harmless environmental substances known as allergens. Type I hypersensitivity reactions are characteristic of allergic reactions and result in the production of excessive amounts of IgE antibodies, which activate basophils and mast cells that cause an inflammatory response. The effects may be systemic, resulting in an anaphylactic reaction, such as vasodilation, mucus secretion, nerve stimulation or smooth muscle contraction, or may be limited to a specific area of ​​the body, for example the respiratory system.

[0003] Phleum pratense Grass allergy is one of the most common forms of allergy that sensitizers suffer from during certain seasons. During the months of late spring and early summer, grass pollen is present in the air, which can cause allergic rhinitis, allergic conjunctivitis and asthma. Direct skin contact with grass, either by sitting on grass or mowing the lawn, can also cause itchy skin, urticarial and atopic dermatitis. One of the most representative species of grass is Phleum pratense. At least nine different allergens have been identified in Phleum pratense species. These are Phl p 1 (Beta-expansin, 27 kDa); Phl p 2 (Grass group II / III, 10-12 kDa); Phl p 4 (55 kDa protein), Phl p 5 (32 kDa), Phl p 6 (11 kDa), Phl p 7 (Calcium-binding protein, 6 kDa), Phl p 11 (Ole e 1-related protein, 20 kDa), Phl p 12 (Profilin, 14 kDa), and Phl p 13 (Polygalacturonase, 55 kDa).

[0004] Olive (Olea europaea) Olive pollen allergy is an important health problem in the Mediterranean region due to the extensive cultivation of olive trees. Olive trees release large amounts of pollen into the air. Currently, 12 allergens from olive (O. europaea) have been described by the WHO / IUIS Allergen Nomenclature Sub-Committee; 11 are from the pollen and one (thaumatin, also known as Ole e 13) is a food allergen from the olive fruit. Ole e 1 is the major allergen and is recognized by more than 70% of olive pollen-sensitized patients. Other allergens include profilin (Ole e 2), porcalcin (Ole e 3 and Ole e 8), glucanase (Ole e 4 and Ole e 9), superoxide dismutase (Ole e 5), lipid transfer protein (Ole e 7), glycosyl hydrolase (Ole e 10), pectin methylesterase (Ole e 11) and Ole e 6.

[0005] House dust mite (Dermatophagoides pteronyssinus) Dust mites are the main source of sensitization worldwide, especially in areas where humidity and temperature are favorable for their development. Currently, the house dust mite (HDM) belonging to the Pyroglyphidae family is the most abundant mite living indoors and is therefore the main source of indoor allergens. Nineteen allergens have been described in D. pteronyssinus by the WHO / IUIS Subcommittee on Allergen Nomenclature, nine of which are proteases and have activities related to allergenicity. The most important allergens are Der p 1 (a 25 kDa cysteine ​​protease) and Der p 2 (a 14 kDa protein of the NPC2 family).

[0006] Allergy sufferers may be treated with drugs to reduce symptoms and control the peak of symptoms, or with specific immunotherapy. However, specific immunotherapy is the only treatment that has the potential to change the course of the disease. Specific immunotherapy (SIT) involves the administration of gradually increasing doses of allergen extracts with the aim of inducing immune tolerance. Rather than relieving the symptoms induced by the allergic reaction, allergen immunotherapy modulates the immune response to the allergen, which can either reduce the need for medication, reduce the severity of symptoms, or completely eliminate hypersensitivity.

[0007] One of the risks of immunotherapy is that allergen injection in sensitized patients may trigger an allergic reaction or anaphylaxis. Since its first use in the early 20th century, many efforts have been made to further improve the safety and efficacy of allergen immunotherapy. One approach is the development of allergoids, which use allergen vaccines that reduce allergenicity but maintain immunogenicity.

[0008] European patent EP 0 662 080 (CBF LETI SA) discloses a method for removing substances and other low molecular weight substances in order to purify allergen extracts and increase the final allergen / protein content. The method consists of disrupting electrostatic, hydrophobic or other physical forces under conditions that separate non-allergenic compounds from allergen-active proteins. The method may consist of a mild acid treatment by lowering the pH below the pI of each allergen protein.

[0009] One of the various methods to reduce allergenicity consists in chemically modifying natural allergen extracts with aldehydes, mainly formaldehyde and glutaraldehyde, to produce allergoids. This aldehyde treatment results in reaction products (mainly polymers) that lose part of the allergenicity (i.e., show a reduction in IgE-reactive B cell epitopes) and have fewer allergic side effects. At the same time, the natural immunogenicity of the allergen is preserved. This allergen modification route has been chosen by some allergen vaccine manufacturers, and commercial products based on this principle have been developed. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] European Patent No. 0 662 080 Summary of the Invention [Problem to be solved by the invention]

[0011] However, there is still interest in finding further ways to optimize the purification of allergens to ensure the removal of low molecular weight proteins, irritants and toxic components, thereby obtaining safe and effective medicines for use in the immunotherapy of allergic diseases. [Means for solving the problem]

[0012] The inventors have found that depigmenting an allergen extract with base can increase the protein content of the extract as well as the major allergen content and biological potency.

[0013] According to a first aspect of the present invention there is provided a method for producing a decolourised allergen extract, the method comprising the steps of: a) basifying the natural allergen extract; and b) removing molecules having a molecular size of less than 3.5 kDa; and c) Adjust the pH to neutral thereby producing a decolorized allergen extract.

[0014] The method further comprises: d) contacting the decolorized allergen extract with an aldehyde; and e) Removing molecules with a molecular size of less than 100 kDa The method may also include a polymerization step including:

[0015] According to a second aspect of the present invention there is provided a decolourised allergen extract obtainable according to the method of the first aspect of the present invention. According to a third aspect of the present invention there is provided a decolourised polymerised allergen extract obtainable according to the method of the first aspect of the present invention.

[0016] According to a further aspect of the present invention there is provided a purified allergen extract for use as an active therapeutic substance in the treatment of allergies.

[0017] definition An "allergen" may be defined as a molecule capable of inducing an IgE response and / or a type I allergic reaction.

[0018] The term "natural allergen extract" refers to an allergen extract that has been extracted from the source material and then treated to remove unbound low molecular weight components. The term "decolorized allergen extract" as referred to in this specification can be defined as a semi-purified allergen extract obtained by removing allergenically irrelevant substances bound to the allergen proteins, including adsorbed pigments, from the natural allergen extract.

[0019] The term "bleached polymerized allergen extract" as referred to in this specification can be defined as a purified allergen extract in which no protein bands of <100 kDa are detectable by non-reducing SDS-PAGE and which is obtained by polymerizing the bleached allergen extract. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The allergen extract of the present invention is derived from any source material containing natural allergens known to induce IgE-mediated immune responses in individuals. Such allergens may include air-borne allergens (e.g., pollen from grasses, trees, herbs and weeds, dust mites, fungi and molds), food allergens (e.g., peanuts), insect allergens (e.g., from cockroaches and fleas, and bee and wasp venoms), and epidermal allergens (animal hair and animal dander, e.g., cat and dog dander).

[0021] The source material can be any allergen, including food allergens (e.g., peanuts), airborne allergens (e.g., pollens (such as tree pollen, weed pollen, grass pollen, and cereal pollen), dust mites, fungi, and molds), epithelial allergens (animal hair and dander, e.g., cat hair and dander, and dog hair and dander), and insect allergens (e.g., from cockroaches and fleas, and bee and wasp venom).

[0022] Pollen allergens from trees, grasses and weeds are derived from taxonomic order groups such as Fagales (e.g., the genera Alnus and Betula), Lamiales (e.g., the genera Olea and Plantago), Poales (e.g., Phleum pratense), Asterales (e.g., the genera Ambrosia and Artemisia), Cayophyllales (e.g., Chenopodium and Salsola), Rosales (e.g., the genus Parietaria), Proteales (e.g., the genus Platanus), etc. Dust mites belong to the order group Astigmata (e.g. Dermatophagoides and Euroglyphus). Airborne allergens derived from molds and fungi belong to the orders Pleosporales (e.g. Alternaria) and Capnodiales (e.g. Cladosporium).

[0023] Airborne allergens include tree pollens (Alnus glutinosa, Betula alba, Corylus avellana, Cupressus arizonica, Olea europaea, Platanus sp.), grass pollens (Cynodon dactylon, Dactylis glomerata, Festuca elatior, Holcus lanatus, Lolium perenne, Phleum pratense, Phragmites communis, Poa pratensis), and weed pollens (Ambrosia elatior, mugwort (Artemisia vulgaris), lamb's paw (Chenopodium album), wall nettle (Parietaria judaica), plantain (Plantago lanceolata), hijiki (Salsola kali)) and cereal pollen (oats (Avena sativa), barley (Hordeum vulgare), rye (Secale cereale), bread wheat (Triticum aestivum), maize (Zea mays)), dust mites (Dermatophagoides farinae, Dermatophagoides microceras, Dermatophagoides pteronyssinus, Euroglyphus maynei), storage mites (Acarus siro), Blomia tropicalis, Lepidoglyphus destructor, Tyrophagus putrescentiae, Glycyphagus domesticus, Chortoglyphusarcuatus) and fungi and molds (Alternaria alternata, Cladosporium herbarum, Aspergillus fumigatus).

[0024] The epithelial allergen may be selected from any animal including cat hair and dander, dog hair and dander, horse hair and dander, human hair and dander, rabbit hair and dander, rat hair and dander, mouse hair and dander, guinea pig hair and dander and feathers.

[0025] Arthropod allergens may be selected from insects, such as ant, flea, cockroach, wasp and bee venom, or mites (Acarina officinalis, Dermatophagoides tropicalis, Dermatophagoides farinae, Dermatophagoides microceras, Dermatophagoides pteronyssinus, Pale-legged mite, Tyrophagus putrescentiae and Cortoglyphus alquatus).

[0026] Pollen allergens respond particularly well to base treatment. Pollen allergens include tree pollens, weed pollens, grass pollens and cereal pollens derived from taxonomic order groups such as Fagales (e.g., Alder and Betula), Lamiales (e.g., Olea and Plantago), Poales (e.g., Timothyrium), Asterales (e.g., Ragweed and Artemisia), Caryophyllales (e.g., Chenopodium and Lamium), Rosales (e.g., Lycoris), and Proteales (e.g., Platanus).

[0027] In one embodiment, the allergen extract is derived from a source material that is pollen. The pollen may be selected from Timothy grass, olive and birch (Betula pendula). More preferably, the source material is selected from peanut (Arachis hypogaea), pollen (timothyme, olive and birch), mites (Dermatophagoides pteronyssinus), and cuticle (cat dander).

[0028] In a preferred embodiment of the invention, the source material is selected from pollen (timothy grass, olive and birch (Betula gracilis)) and mites (Dermatophagoides pteronyssinus). More particularly, the raw material is selected from Timothy grass, Olive and White birch (Betula medica).

[0029] In a further preferred embodiment of the invention, the source material is Timothy grass. Alternatively, the source material is olives. Alternatively, the source material is Dermatophagoides pteronyssinus.

[0030] One method for producing a natural allergen extract is described below, however it will be appreciated that other suitable methods for obtaining a natural allergen extract will be known to those skilled in the art and may be used as a starting material in the decolorization process of the present invention.

[0031] The method for obtaining natural allergen extracts from raw materials includes: i) contacting the source material or a first source material residue with a liquid allergen extractant to produce a second mixture of dissolved allergens in a liquid phase and a solid phase comprising a second source material residue; ii) subjecting the second mixture to a second separation step to isolate the allergens dissolved in the liquid phase and produce a crude allergen extract; iii) subjecting the crude allergen extract to a low molecular weight fraction removal step to remove molecules having a size of less than 1-10 kDa, preferably less than 3.5 kDa, at 3-10°C; iv) carrying out step iii) at 3-10°C until the allergen extract has a conductivity of less than 900 μS / cm measured at room temperature to obtain a natural allergen extract. This may include:

[0032] The source material may first be treated to create a maximum surface area for contact with the liquid allergen extractant. The source material may be homogenized, blended, ground, or powdered to produce a uniform slurry for liquid extraction.

[0033] In some cases, a preliminary degreasing step may be necessary. i) contacting an allergen-containing source material with a liquid lipid extractant to produce a first mixture containing lipids dissolved in a liquid phase and a solid phase consisting of the first source material residue containing allergens and proteins; and ii) subjecting the first mixture to a first separation step to isolate a first source material residue. This may include:

[0034] A preliminary defatting step may be necessary when the source material is pollen, epidermis, or foodstuffs, e.g. nuts such as peanuts. The lipid extraction, or "defatting" step, removes lipophilic compounds such as lipids and fatty acids from the source material.

[0035] The liquid lipid extractant can be acetone, ether, or a similar solvent, which can be at low temperature. Preferably, the liquid lipid extractant is acetone. The lipid extraction process can be carried out in a 1:1 weight ratio of raw material to liquid lipid extractant, or in any ratio where the weight of the liquid lipid extractant exceeds the weight of the raw material, for example, 1:2, 1:3, 1:5, 1:10. The lipid extraction process is preferably carried out in a ratio of 1 kg of raw material to 2 L of liquid lipid extractant. The lipid extraction process is preferably carried out for a time sufficient to dissolve the lipids in the raw material in the liquid lipid extractant, which can be more than 1 minute, preferably more than 5 minutes, more preferably more than 30 minutes, and most preferably more than 1 hour. The liquid extraction process can be carried out at a temperature between 2 and 25°C, but is preferably carried out at a low temperature between 2 and 6°C, more preferably 3 and 5°C. During the lipid extraction process, the raw material is preferably stirred or agitated with the liquid lipid extractant.

[0036] The first separation step can be any suitable separation step known to those of skill in the art, for example, the first separation step can be filtration or any other suitable method. After the first separation step, the raw material residue can be washed with a liquid lipid extractant. Optionally, the first raw material residue can be further extracted with a liquid lipid extractant and then separated. Preferably, one, two or more additional lipid extraction steps are carried out. The filtration of the liquid extractant can be repeated until it is clear. Other suitable separation methods known to those skilled in the art can also be used.

[0037] After lipid extraction and separation, the raw material residue can be dried. The raw material residue can be dried at 2-25° C., preferably at room temperature. The drying process preferably continues for a sufficient time to allow the liquid lipid extractant to be removed from the raw material residue. This can be 1-24 hours, 6-18 hours, but drying is preferably at least 12 hours.

[0038] Allergens can be obtained from the ("defatted") first raw material residue by extraction with a liquid allergen extractant to produce a crude allergen extract with the allergens dissolved in the liquid phase and a solid phase consisting of a "waste" non-allergenic residue. The liquid allergen extractant may be an aqueous solution, preferably comprising a buffer. The liquid allergen extractant may comprise PBS and / or NaCl. For example, it may be an aqueous solution of 0.01M PBS / 0.15M NaCl, or ammonium bicarbonate ((NH 4 )HCO 3 ) and / or an aqueous solution of NaCl, e.g. 0.125M (NH 4 )HCO 3The liquid allergen extractant may be an aqueous solution of 0.15M NaCl / 0.15M. The first source material residue may be extracted into the liquid allergen extractant in any ratio where the weight of the liquid allergen extractant exceeds the weight of the first source material residue, for example, 1:2, 1:3, 1:5, 1:10, 1:20, 1:50, 1:80 by weight. Preferably, the first source material residue is extracted into the liquid allergen extractant in a first source material residue:liquid allergen extractant weight ratio of 1:10. The ratio of the first source material residue to the liquid allergen extractant may vary, but should be such that the allergens in the first source material residue are soluble in the liquid allergen extractant. The extraction of the first source material residue with the liquid allergen extractant is preferably carried out for a time sufficient for the allergens in the first source material residue to be dissolved in the liquid allergen extractant. It may be 30 minutes to 12 hours, preferably 1 to 6 hours, more preferably 2 to 5 hours, and most preferably about 4 hours. The allergen extraction step may be carried out at a temperature of 2 to 25° C., but is preferably carried out at a low temperature of 2 to 6° C., more preferably 3 to 5° C. During the allergen extraction step, the first raw material residue is preferably stirred or agitated with the liquid allergen extractant.

[0039] After the allergen extraction step, the allergens dissolved in the liquid phase can be separated from the second raw material residue to produce a crude allergen extract. The separation step is preferably centrifugation, but many techniques for separating solids from liquids are applicable and are well known to those skilled in the art. Preferably, the allergens dissolved in the liquid phase are centrifuged at 2-6°C, preferably 3-5°C, for a time sufficient for the raw material residue to settle as a pellet, for example, 1 min to 1 h, or more than 1 h. The crude allergen extract (i.e., the supernatant containing the dissolved allergens) can be stored at 2-6°C. The second raw material residue pellet may be further extracted with a liquid allergen extractant using the same conditions as the first allergen extraction step, preferably for a longer extraction time, for example, 4-8 h, 8-12 h, or more than 12 h. After the second allergen extraction step, the allergens dissolved in the liquid phase can be separated from the second raw material residue to produce a crude allergen extract. The crude allergen extracts from the first and second allergen extraction steps are preferably pooled for further processing.

[0040] The crude allergen extract can be filtered, for example, using a pore size of 0.45 μm. The crude allergen extract can be subjected to a low molecular weight fraction removal step to remove molecules with a low molecular weight size, such as salts and other non-allergenic compounds. In step iii), molecules with a molecular weight size of less than 1-10 kDa, preferably less than 3.5 kDa, can be removed. The low molecular weight fraction removal step is preferably continued at 3-10° C., preferably 3-5° C., until the conductivity of the allergen extract is less than 900 μS / cm, or less than 800 μS / cm, or less than 700 μS / cm, or less than 600 μS / cm, or even more preferably less than 500 μS / cm (measured at room temperature).

[0041] The obtained natural allergen extract can be filtered, for example, using a pore size of 0.45 and / or 0.22 μm. Natural allergen extracts can be used for the preparation of pharmaceutical compositions or vaccines for standardization, diagnostic, synthesis and vaccination purposes.

[0042] In the present invention, a natural allergen extract is used as the starting material for the decolorization process described herein. The present invention provides a decolorization method that includes a basification treatment to remove non-allergenic compounds attached to allergens / proteins using a means to disrupt the electrostatic, hydrophobic or other physical forces that cause the non-allergenic compounds to adhere to the protein, thereby producing a decolorized extract.

[0043] The basic treatment is a) basifying the natural allergen extract; b) removing molecules having a molecular size of less than 3.5 kDa; and c) Adjust the pH to neutral to produce a decolorized allergen extract. This includes:

[0044] The basification treatment includes either a weak base treatment or a strong base treatment. With base treatment, the pH of the allergen / protein may be increased to at least pH 7, for example to a pH value between 7 and 11. The preferred pH for allergen proteins is between 7 and 10, more preferably between 7 and 8. A pH value higher than 11 may result in an incomplete protein profile of the decolorized allergen extract, and a neutral pH, for example pH 6, may result in an incomplete removal of non-allergenic compounds in the resulting decolorized allergen extract.

[0045] In one embodiment, the base treatment comprises basifying the allergen extract to pH 7, pH 8, pH 9, pH 10 or pH 11. In one embodiment, the base treatment comprises basifying the allergen extract to a pH of 7-11, or a pH of 7-10, preferably a pH of 7-8.

[0046] The pH of the natural allergen extract can be increased using any suitable base. The base can be a strong or weak base. Strong bases include sodium hydroxide, lithium hydroxide and potassium hydroxide. Weak bases include urea, ammonium hydroxide and methylamine. In one embodiment, the base is selected from the list including sodium hydroxide, lithium hydroxide, potassium hydroxide, urea, ammonium hydroxide or methylamine. In particular, the base is sodium hydroxide.

[0047] The basified extract may be maintained at the basic pH for between 1 minute and 24 hours, between 1 minute and 4 hours, between 1 and 60 minutes, preferably between 5 and 30 minutes, more preferably between 10 and 20 minutes, and most preferably for approximately 15 minutes.

[0048] Molecules having a molecular size of less than 3.5 kDa can be removed in a low molecular weight fraction removal step. After the basification treatment, the resulting decolorized allergen extract is recovered and the pH of the extract can be adjusted using a suitable acid, for example HCl, to a value that avoids protein precipitation, for example pH 7.0-7.5, more particularly pH 7.3-7.4.

[0049] In particular, the basic treatment is a) basifying a natural allergen extract to a pH of 7 to 11, and maintaining the basified extract for 1 minute to 24 hours, for example, 5 to 30 minutes, and preferably 15 minutes; b) subjecting the extract to a low molecular weight fraction removal step to remove molecules having a molecular size of less than 3.5 kDa; and c) Adjust the pH to 7.0-7.5, especially 7.3-7.4, to produce a decolorized allergen extract. This may include:

[0050] The basification process may involve basifying the natural allergen extract to a pH of 7-10. The low molecular weight fraction removal step can be a dialysis step in which the extract is dialyzed against a dialysate such as purified water or a buffer solution. The low molecular weight fraction removal step can be carried out at 2-25° C., but is preferably carried out at a lower temperature of 2-6° C., and most preferably 3-5° C. The low molecular weight fraction removal step can be carried out for 12-24 hours, in which case the solvent, or the dialysate in the case of dialysis, is replaced periodically to maintain the reaction.

[0051] The resulting decolorized allergen extract can be filtered, for example using 0.45 μm and / or 0.22 μm pore size, and frozen or lyophilized for storage. The extract produced using the method of the present invention can be further processed. d) contacting the decolorized allergen extract with an aldehyde and, after polymerization, e) Removing molecules with a molecular size of less than 100 kDa The polymerization step may include

[0052] The aldehyde may be any suitable aldehyde, for example glutaraldehyde or formaldehyde. The polymerization process is d) contacting the decolorized allergen extract with glutaraldehyde or formaldehyde; e) subjecting the extract to a molecular fraction removal step to remove molecules having a molecular size of less than 100 kDa; and f) Step e) is carried out at 3-15°C, preferably 3-5°C, until the allergen extract has a conductivity of less than 210 μS / cm (measured at room temperature) and / or until the extract is free of glutaraldehyde, to obtain a decolorized polymerized allergen extract. This may include:

[0053] If the extract for polymerization is lyophilized, it can be reconstituted in a buffer, e.g., 0.01M PBS / 0.15M NaCl, to a final concentration of 0.1-500 mg / ml, preferably 1-100 mg / ml, most preferably 10-50 mg / ml.

[0054] The polymerization reaction is preferably carried out to completion such that no protein bands of <100 kDa (eg, 14-25 kDa) are detectable in the bleached polymerized allergen extract by non-reducing SDS-PAGE.

[0055] Increasing the glutaraldehyde concentration may result in a lower polymer yield and an increased residue yield resulting from any centrifugation step prior to dialysis. For known polymerization conditions using a glutaraldehyde concentration of approximately 5 mg / ml (i.e. 0.009 ml glutaraldehyde per ml allergen extract), the optimal glutaraldehyde concentration was experimentally determined to be approximately twice the known amount for some allergens (peanut, cat scutellaria and ragweed), i.e. 10 mg / ml (0.02 ml glutaraldehyde per ml allergen extract). Aldehydes can be added in the range of 1-20 mg / ml. Although it is possible to use a known amount of final glutaraldehyde concentration to result in partial polymerization of the allergen, to achieve optimal polymerization, the aldehyde is preferably added at a final concentration of 5-10 mg / ml or in a ratio of 0.01-0.02 ml glutaraldehyde per ml of extract.

[0056] Reducing the rate of glutaraldehyde addition may also result in a lower polymer yield and an increased residue yield. The aldehyde may be added to the extract at a constant rate, for example, 0.001-0.5 ml / min (1-500 μl / min or 60-3000 μl / hr).

[0057] The polymerization reaction may be maintained at room temperature or higher for 1-12 hours, preferably 7 hours. The polymerization reaction may be stopped with glycine at a ratio of 40 mg per ml of decolorized polymerized allergen extract solution. The stopped reaction may be maintained at 3-5°C overnight, preferably under stirring. The decolorized polymerized allergen in the liquid phase may be separated from the insoluble residue to produce the decolorized polymerized allergen extract. The separation step is preferably centrifugation, but many separation techniques are applicable and are well known to those skilled in the art. Preferably, the extract is centrifuged at 2-6°C, preferably 3-5°C, for a time sufficient for the insoluble residue to settle as a pellet, for example 1 min to 1 h, or more than 1 h. The supernatant (containing the soluble decolorized polymerized allergen) is collected and may be subjected to a molecular fraction removal step e).

[0058] In step e), molecules with a molecular size below 100 kDa are removed. Preferably, the molecular fraction removal step is a dialysis step in which the extract is dialyzed against a dialysate such as purified water or a buffer solution at 3-15° C., preferably 3-5° C. The molecular fraction removal step can be continued at 3-15° C., preferably 3-5° C., until the conductivity measured at room temperature is less than 300 μS / cm, more preferably 250 μS / cm, most preferably less than 210 μS / cm.

[0059] The resulting decolorized polymerized allergen extract can be filtered, for example using 0.45 μm and / or 0.22 μm pore size, and frozen or lyophilized for storage. Any of the small molecule fraction removal steps described herein, such as steps b) or e), may comprise an ultrafiltration step, a diafiltration step, a dialysis step, or a filtration step.

[0060] In its simplest form, the method of the present invention may comprise producing or obtaining a natural allergen extract, and basifying the extract, for example by weak or strong base treatment, to remove non-allergenic compounds with low molecular size.The extract can then be polymerized with an aldehyde.The natural allergen extract may be a peanut, pollen, grass, cuticle, mold, fungus, insect or mite allergen, particularly a grass, pollen or mite, more particularly a pollen allergen.The method of the present invention results in an allergen extract that exhibits reduced IgE binding capacity, but retains its immunogenic capacity.

[0061] The present invention further includes a treatment for allergy and a diagnostic agent for allergy, both of which contain as an active ingredient the allergen extract produced by the method of the present invention. Allergy can be associated with exposure to various allergens that cause an IgE-mediated allergic response, as discussed herein.

[0062] According to a second aspect of the present invention there is provided a decolourised allergen extract obtainable according to the method of the first aspect of the present invention. According to a third aspect of the present invention there is provided a decolourised polymerised allergen extract obtainable according to the method of the first aspect of the present invention. Also provided is a purified allergen extract for use as an active therapeutic substance.

[0063] The allergen extract may be selected from peanut (Arachis hypogaea), pollen (Phleum pratense, Betula alba, Olive (Olea europaea), Wall nettle (Parietaria judaica, and Arizona cypress (Cupressus arizonica)), dust mite (Dermatophagoides pteronyssinus), and cuticle (cat dander). In particular, the allergen extract is pollen selected from olive and timothy grass.

[0064] The allergen extract may be for use in the treatment of allergies. In a preferred embodiment, the olive or timothy grass allergen extract may be for use in the treatment of pollen allergies.

[0065] The decolorized polymerized allergen extract may be characterized by the following physicochemical and biological properties: i. soluble in water, ii. Absence of non-polymerized allergens / proteins with molecular weights less than 100 kDa (as identified by bands on SDS-PAGE under non-reducing conditions) iii. Absence of IgE-recognition bands with molecular weights less than 100 kDa (confirmed by immunoblotting under non-reducing conditions) iv. Absence of polymeric molecules with molecular weight less than 100 kDa (as determined by SDS-PAGE) v. Reduced biological potency (95%) against natural allergen extracts (as determined by IgE ELISA inhibition experiments using defined serum pools of sensitized individuals); and vi. No abnormal toxicity in mice and guinea pigs In particular, the decolorized polymerized allergen extract is characterized by a reduced biological potency (95%) relative to the natural allergen extract (as determined by IgE ELISA competition experiments using defined serum pools of sensitized individuals).

[0066] The allergen extract of the present invention may be for use as an active ingredient of a medicine for the treatment of allergy sufferers, with the aim of inducing tolerance to a particular allergen.

[0067] The use of the allergen extract according to the invention is provided for the diagnosis of immunological disorders, preferably for detecting allergic diseases. The use of the allergen extract according to the invention is provided for the treatment of allergies or for the manufacture of a medicament for the treatment of allergies, for example pollen allergies. The use may be for immunotherapy. The use may be for standardization, diagnostic, synthesis and vaccination purposes. The use may be in the therapeutic treatment of patients, in particular in immunotherapy. The use may be in the monitoring of patients during immunotherapy.

[0068] Alternatively, there is provided a method for treating a patient in need of treatment for an allergy, such as a pollen allergy, comprising the step of administering to a patient in need thereof the allergen extract of the present invention.

[0069] According to further aspects of the present invention, there is provided a pharmaceutical composition comprising an allergen extract according to the present invention, a pharmaceutical composition for the treatment of allergy comprising as an active ingredient a pharma- ceutical effective amount of an allergen extract according to the present invention and at least one pharma- ceutical acceptable carrier or diluent, and a diagnostic composition for allergy comprising as an active ingredient a diagnostically effective amount of an allergen extract according to the present invention.

[0070] According to a further aspect of the present invention, there is provided a vaccine comprising the allergen extract according to the present invention. The pharmaceutical composition and vaccine may further comprise one or more adjuvants, diluents, preservatives or mixtures thereof. The pharmaceutical composition or vaccine may comprise a physiologically acceptable carrier. As used herein, the term "pharmaceutical acceptable" preferably means approved by a government regulatory agency or listed in the European or United States Pharmacopoeia or another generally recognized pharmacopoeia for human use.

[0071] Such pharma- ceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients are mannitol, human serum albumin (HSA), starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium carbonate, magnesium stearate, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like.

[0072] There is provided a vaccine obtainable according to the method of the first aspect of the invention. The vaccine may be subcutaneous or sublingual or transdermal. There is provided the use of a vaccine according to the invention in the treatment of allergy, or in the manufacture of a medicament for the treatment of allergy.

[0073] According to a further aspect of the present invention there is provided a method of preventing allergen sensitisation comprising exposing an individual to an effective amount of the allergen extract, pharmaceutical composition or vaccine of the present invention.

[0074] According to a further aspect of the present invention there is provided a method for treating allergy in a sensitized individual, the method comprising administering to the individual an effective amount of an allergen extract, pharmaceutical composition or vaccine of the present invention, which may be administered subcutaneously or sublingually and may be administered as increasing or fixed doses.

[0075] The individual may be a human or an animal, preferably a human. [Brief description of the drawings]

[0076] [Figure 1]Figure 1 shows the protein content (measured using the Lowry-Biuret method) of Timothy grass extracts of freeze-dried samples obtained after different pH treatments. Error bars indicate the standard deviation of the mean values ​​of the different samples. [Diagram 2] Figure 2 shows the protein content (measured using the Lowry-Biuret method) of Timothy grass extract of freeze-dried samples obtained after treatment with different bases. Error bars indicate the standard deviation of the mean values ​​of the different samples. [Diagram 3] Figure 3 shows the Phl p 5 (major allergen in Timothy grass) content of freeze-dried samples obtained after different pH treatments. Error bars indicate the standard deviation of the mean values ​​of the different samples. [Figure 4] Figure 4 shows the Phl p 5 (major allergen in Timothy grass) content of the freeze-dried samples obtained after treatment with different bases. The error bars indicate the standard deviation of the mean values ​​of the different samples. [Diagram 5] Figure 5 shows the biological potency (ELISA competition) of Timothy grass extract of lyophilized samples obtained after different pH treatments. Error bars indicate the standard deviation of the mean values ​​of the different samples. [Figure 6] Figure 6 shows the biological potency (ELISA competition) of Timothy grass extract of lyophilized samples obtained after treatment with different bases. Error bars indicate the standard deviation of the mean values ​​of the different samples. [Figure 7] Figure 7 shows the number of µg required to obtain 50% inhibition of IgE binding to the native extract of Timothy grass extract of lyophilized samples obtained after different pH treatments. Error bars indicate the standard deviation of the mean values ​​of the different samples. [Figure 8] Figure 8 shows the number of μg required to obtain 50% inhibition of IgE binding to the native extract of Timothy grass extract of lyophilized samples obtained after treatment with different bases. Error bars indicate the standard deviation of the mean values ​​of the different samples. [Figure 9] FIG. 9 shows the SDS of Timothy grass extracts treated with different bases. [Figure 10]FIG. 10 shows a Western blot of Timothy grass extracts treated with different bases. [Figure 11] Figure 11 shows the protein content of olive extracts measured using the Lowry-Biuret method for freeze-dried samples obtained after treatment with different pH values. Error bars indicate the standard deviation of the mean values ​​of the different samples. [Figure 12] Figure 12 shows the protein content of olive extracts measured using the Lowry-Biuret method of freeze-dried samples obtained after treatment with different bases. The error bars indicate the standard deviation of the mean values ​​of the different samples. [Figure 13] Figure 13 shows the biological potency of olive extract measured using the competitive ELISA method of lyophilized samples obtained after treatment with different pH values. Error bars indicate the standard deviation of the mean values ​​of the different samples. [Figure 14] Figure 14 shows the biological potency of olive extracts measured using the competitive ELISA method of lyophilized samples obtained after treatment with different bases. Error bars indicate the standard deviation of the mean values ​​of the different samples. [Figure 15] Figure 15 shows the number of μg required to obtain 50% inhibition of IgE binding to olive oil native extract of lyophilized samples obtained after treatment with different pH. Error bars indicate the standard deviation of the mean values ​​of the different samples. [Figure 16] Figure 16 shows the number of μg required to obtain 50% inhibition of IgE binding to the olive oil native extract of lyophilized samples obtained after treatment with different bases. Error bars indicate the standard deviation of the mean values ​​of the different samples. [Figure 17] FIG. 17 shows the SDS of olive extracts treated with different bases. [Figure 18] FIG. 18 shows a Western blot of olive extracts treated with different bases. [Figure 19]Figure 19 shows thin layer chromatography of olives. A, samples treated with sodium hydroxide and lithium hydroxide; B, treatment with ammonium hydroxide, sodium hydroxide and urea; C, treatment with methylamine. All assays were compared to the natural extract. Standards were: 1, chlorogenic acid; 2, quercetin; 3, rutin trihydrate; 4, isoquercitrin; 5, quercitrin; 6, kaempferol 3-glucoside; 7, apigenin 7-glucoside. [Figure 20] Figure 20 shows the protein content of Dermatophagoides pteronyssinus extract measured using the Lowry-Biuret method of freeze-dried samples obtained after treatment with different pH. Error bars indicate the standard deviation of the mean values ​​of the different samples. [Figure 21] Figure 21 shows the protein content of Dermatophagoides pteronyssinus extract measured using the Lowry-Biuret method of freeze-dried samples obtained after treatment with different bases. Error bars indicate the standard deviation of the mean values ​​of the different samples. [Figure 22] Figure 22 shows the main allergen content of Dermatophagoides pteronyssinus extract of the lyophilized samples obtained after treatment with different pH, determined using specific ELISA sandwich kits (indoor) for Der p 1 and Der p 2. Error bars indicate the standard deviation of the mean values ​​of the different samples. [Diagram 23] Figure 23 shows the main allergen content of Dermatophagoides pteronyssinus extract of lyophilized samples obtained after treatment with different acids or bases, determined using specific ELISA sandwich kits (indoor) for Der p 1 and Der p 2. Error bars indicate the standard deviation of the mean values ​​of the different samples. [Figure 24] Figure 24 shows the biological potency of Dermatophagoides pteronyssinus extract measured using the ELISA competitive method of lyophilized samples obtained after treatment with different pH. Error bars indicate the standard deviation of the mean values ​​of the different samples. [Diagram 25]Figure 25 shows the biological potency of Dermatophagoides pteronyssinus extract measured using the ELISA competitive method of lyophilized samples obtained after treatment with different bases. Error bars indicate the standard deviation of the mean values ​​of the different samples. [Figure 26] Figure 26 shows the biological potency of Dermatophagoides pteronyssinus extract measured using the ELISA inhibition method (μg required to obtain 50% inhibition of IgE binding to the native extract) of lyophilized samples obtained after treatment with different pH. Error bars indicate the standard deviation of the mean values ​​of the different samples. [Figure 27] Figure 27 shows the biological potency of Dermatophagoides pteronyssinus extract measured using the ELISA inhibition method (number of μg required to obtain 50% inhibition of IgE binding to the native extract) of lyophilized samples obtained after treatment with different bases. Error bars indicate the standard deviation of the mean values ​​of the different samples. [Figure 28] FIG. 28 shows the SDS of Dermatophagoides pteronyssinus extract treated with different bases. [Figure 29] FIG. 29 shows a Western blot of Dermatophagoides pteronyssinus extract treated with different bases. EXAMPLES

[0077] The invention is illustrated by the following examples, which detail the methods of preparation, purification and basification of allergen-containing extracts. Methods A to C detail the methods used to prepare the allergen extracts.

[0078] method A. Optimal degreasing method for allergen raw materials A defatted extract was obtained. Generally, the homogenized material was defatted in acetone at 3-5°C and filtered. This process was repeated until the acetone was clear. The defatted material was collected and dried at room temperature until all the acetone was removed.

[0079] B. Production of natural allergen extracts The dried defatted material was weighed and extracted in 0.01M PBS / 0.15M NaCl in a ratio of 1:10 for 4 hours at 3-5°C under magnetic stirring. The solution was then centrifuged for 30 minutes at 4°C and 10,000 rpm. The resulting supernatant was collected and stored at 3-5°C. The pellet was reconstituted in 0.01M PBS / 0.15M NaCl (1:10) and extracted overnight at 3-5°C under magnetic stirring. The solution was centrifuged for 30 minutes at 3-5°C and 10,000 rpm, and the supernatant was collected and mixed with the previously obtained fraction. The combined extract was filtered using a pore size of 0.45 μm and dialyzed extensively with a 3 kDa cut-off dialysis membrane until the conductivity was less than 500 μS / cm. The extract was then filter-sterilized using a pore size of 0.22 μm.

[0080] C. Preparation of decolorized allergen extract The natural extract, which had been kept in aqueous solution at 3-5°C, was further processed using the following procedure. Under magnetic stirring, the pH of the solution was adjusted to pH 7-11 by the addition of sodium hydroxide, lithium hydroxide, potassium hydroxide, urea, ammonium hydroxide or methylamine and maintained under these conditions for 15 minutes. The extract was then dialyzed with purified water in a 3.5 kDa cut-off dialysis membrane for approximately 17 hours at 3-5°C against 10 volumes of purified water. During this time, the purified water was replaced four times. After base treatment, the extract was collected and the pH was adjusted to 7.3-7.4 using 0.1 M HCl. Finally, the extract was sterile filtered to 0.22 μm, frozen and lyophilized.

[0081] Immunological characterization Protein content The protein content of the native and decolorized extracts was determined by the Lowry Biuret method according to the manufacturer's instructions.

[0082] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Protein profiles were confirmed by SDS-PAGE on acrylamide-acrylamide gels at 2.67% C, 15% T under reducing conditions (samples were incubated with β-mercaptoethanol and heated at 95°C for 10 min). Samples and low molecular weight standards (BioRad Laboratories, Hercules, CA, USA) were run on the same gel. Gels were stained with 0.1% Coomassie Brilliant Blue R-250 (BioRad).

[0083] Immunoblot Electrophoretically separated proteins (by SDS-PAGE) were transferred to PVDF membranes (Trans-Blot® Turbo™ Transfer Pack, BioRad) and incubated overnight with serum from patients sensitized to each allergen (Plasmalab International, Everett, WA, USA) diluted in 0.01 M phosphate buffered saline (PBS)-0.1% Tween. Plasmalab International is in full compliance with Food and Drug Administration regulations. Specific IgE binding to the extracts was detected with a peroxidase-conjugated monoclonal antibody, anti-human-IgE-PO (Ingenasa, Madrid, Spain) and stained with luminol solution (Western Immun-Star). TM The expression was performed using a Western CTM Kit (Bio-Rad) and detection was performed by chemiluminescence (ChemiDoc XRS, Bio-Rad).

[0084] Major allergen quantification The major allergens were quantified by ELISA sandwich method using Enzyme-Linked Immunosorbent Assay Detection Kit (Indoor Biotechnologies, VA, USA). Briefly, Nunc Maxisorp plates (Thermo Scientific, Waltham, MA, USA) were coated with specific monoclonal antibodies diluted in carbonate / bicarbonate buffer (pH 9.6) and incubated overnight at 4°C. Afterwards, the plates were blocked with BSA 1% in PBS 0.01M-Tween 0.05%. Samples and standards were then added in half-fold serial dilutions in BSA 1% in PBS 0.01M-Tween 0.05%. Specific secondary monoclonal antibodies (biotinylated) were added and finally streptavidin was used. After stopping with sulfuric acid, the reaction with the color-developing solution (chromogen) was measured at OD 450 nm. Standard curves were obtained using a 4-parameter logistic fit by least squares method, into which the sample concentrations were interpolated to obtain the results.

[0085] ELISA competition (IgE) The ability of native and decolorized extracts to inhibit IgE binding to biologically standardized in-house reference preparations (IHRPs) was compared. Nunc plates (Thermo Scientific) were coated with anti-IgE. A pool of sera from allergen-sensitized patients was incubated on the plates. Dilutions of samples and IHRPs were incubated with peroxidase-labeled allergens. The mixtures were added to the coated plates and incubated. Afterwards, a color development solution (chromogen) was added, stopped with sulfuric acid, and the optical density (OD) was measured at 450 nm.

[0086] ELISA Inhibition (IgE) The in vitro allergenic activity of the extracts (natural and decolorized) was tested by ELISA inhibition, and the 50% inhibition point was established using the natural extract as a reference. Plastic microtiter plates (Immulon 4HBX; Thermo Scientific) were coated overnight with the natural extract (10 μg protein / ml). Serial 1:2 dilutions were made from the natural and decolorized extracts in Nunc F plates (Thermo Scientific). Each dilution was incubated with the serum pool for 2 h at room temperature. The extract dilutions were then transferred to the native (extract) coated plates and incubated for 2 h. After washing, 100 μl of anti-human IgE peroxidase was added and left at room temperature for 30 min. After washing, the plates were developed (chromogen) for 30 min and stopped with sulfuric acid (1N).

[0087] Thin Layer Chromatography (TLC) Plant flavonoids were used as positive controls. Controls and standards were applied to TLC aluminum sheets silica gel 60F (Merck, Darmstadt, Germany). Ethyl acetate:formic acid:acetic acid:water (100:11:11:27) was used as eluent and developed with a solution of 1% methanolic diphenylboronic acid-β-ethylamino ester followed by 5% ethanolic poly(ethylene glycol)-4000.

[0088] Working Example Example 1: Phleum pratense The decolorized Timothy grass extract was obtained according to method steps A-C.

[0089] Protein content The highest protein content was obtained after treatment with methylammonium at pH 8 (865 μg protein / mg lyophilized extract), whereas the lowest content corresponded to treatment with potassium hydroxide at pH 11 (579 μg protein / mg lyophilized extract) (Table 1). The average value across all pH values ​​was 718 μg protein / mg lyophilized extract.

[0090] The highest protein content corresponded to the pH 8 treatment (mean value 758 μg protein / mg lyophilized extract) and the lowest to pH 9 (694 μg protein / mg lyophilized extract) (Table 2) (Figure 2). Protein contents at all pH values ​​between 7 and 11 were higher than those of the native extract and the sample at pH 6, which is the native pH of the sample (samples were treated the same as the decolorized samples, but without the pH change for basification).

[0091] Concerning the different bases used, the highest protein content was achieved with lithium hydroxide (739 μg protein / mg lyophilized extract) and the lowest with urea (668 μg protein / mg lyophilized extract) (Table 3, Figure 2).

[0092] Major allergen quantification The lowest level corresponded to ammonium hydroxide treatment at pH 10 (13.0 μg Phl p 5 / mg freeze-dried extract) (Table 1). The highest levels corresponded to lithium hydroxide treatment at pH 8, followed by ammonium hydroxide treatment at pH 11 (41.0 and 38.0 μg Phl p 5 / mg freeze-dried extract, respectively). The mean decolorization (samples) was 26.6 μg Phl p 5 / mg freeze-dried extract.

[0093] The highest major allergen content was obtained with treatment at pH 8 (31.5 μg Phl p 5 / mg lyophilized extract) and the lowest with treatment at pH 10 (23.2 μg Phl p 5 / mg lyophilized extract) (Figure 3, Table 2). However, all base treatments resulted in a higher major allergen content than the native extract and the pH 6 sample (untreated).

[0094] The lowest major allergen content was obtained by treatment with sodium hydroxide (24.1 μg Phl p 5 / mg lyophilized extract), and the highest with potassium hydroxide (29.1 μg Phl p 5 / mg lyophilized extract) (Figure 4, Table 3).

[0095] ELISA Competitive (IgE) - Biological Potency The highest biological potency corresponded to the methylammonium treated samples at pH 7 and 9 (3052 and 2909 HEPL / mg lyophilized extract, respectively) (Figs. 5 and 6, Table 1). The lowest value corresponded to the treatment with potassium hydroxide (mean value 984 HEPL / mg lyophilized extract), similar to the native extract (952 HEPL / mg) (Table 3). Differences between ammonium hydroxide and the other groups except methylamine could be detected (potassium hydroxide P = 0.010, Tukey test; urea P = 0.005, sodium hydroxide P = 0.012, and lithium hydroxide P = 0.049, Mann-Whitney). There were also differences between methylamine and potassium hydroxide. The mean value of the decolorized samples was 1685 HEPL / mg lyophilized extract, which was higher than the results obtained for the native extract and the sample at pH 6 (without treatment) (952 and 1262 HEPL / mg lyophilized extract, respectively).

[0096] ELISA Inhibition (IgE) The number of micrograms of lyophilization required to reach 50% inhibition did not correlate with the HEPL / mg values ​​(Pearson Product Moment Correlation, P>0.050).

[0097] The 50% inhibition values ​​at pH 8 were significantly higher than those at pH 11 and 10 (P = 0.030 and P = 0.017, respectively, Mann-Whitney Rank Sum Test) (Fig. 7, Table 2). The lowest values ​​corresponded to potassium hydroxide at pH 11 (0.007 μg), followed by ammonium hydroxide at pH 11 (0.020 μg, respectively) (Table 1). The mean values ​​were 0.102 μg, similar to those of the natural extract and pH 6 samples (0.105 and 0.097 μg, respectively).

[0098] Concerning the bases used, the highest 50% inhibition value corresponded to treatment with urea (mean 0.195 μg) (Figure 8, Table 3). The lowest value was observed with methylamine treatment (mean 0.066 μg). Differences were detected between urea treatment and methylamine, ammonium hydroxide, lithium hydroxide, potassium hydroxide and sodium hydroxide.

[0099] Individual results table

[0100] [Table 1]

[0101] Summary of results analyzed by group

[0102] [Table 2]

[0103] [Table 3]

[0104] For native and untreated samples, standard deviations are not shown since only one sample was analyzed. SDS and Western Blot SDS and Western blots were performed on all destained samples and compared with the native extract (Tithmopogon japonicus).

[0105] All electrophoresis was performed on 15% T acrylamide gels under reducing conditions. All lanes were loaded with the same μg of lyophilized sample (25 μg). Gels were stained with Coomassie R-250. Membranes were incubated with a serum pool (1 / 5 dilution) of patients exhibiting IgE against P. pratense (measured using ELISA). Afterwards, membranes were incubated with α-IgE-PO and developed using chemiluminescence. The SDS results are shown in Figure 9. The Western blot is shown in Figure 10.

[0106] The most intense bands in the native extract were observed at 11, 37 and 31 kDa (in order of intensity). The most important difference observed in the SDS decolorized samples was the decrease in intensity of the high molecular weight bands with increasing pH. However, this effect only resulted in low intensity bands and no bands were completely removed.

[0107] Note: Some bands have been sequenced in the IHRP of Timothy grass. Phl p 5 was identified as a 37 kDa band, Phl p 1 as a 31 kDa band, and Phl p 2 (or 3) and 6 as 12 kDa. These allergens are reported in the IUIS with slightly different molecular weights: Phl p 5 at 32 kDa, Phl p 1 at 27 kDa, Phl p 2 at 10-12 kDa, and Phl p 6 at 11 kDa. Other allergens listed in the IUIS are Phl p 4 and 13 at 55 kDa, Phl p 7 (calcium binding protein) at 6 kDa, Phl p 11 (Ole e 1 related) at 20 kDa, and Phl p 12 (profilin) ​​at 14 kDa.

[0108] Furthermore, Western blot was also performed (Figure 10). The most intense bands in the native extract corresponded to 37, 31, 59, 15 and 12 kDa (in order of intensity), which may correspond to Phl p 5, Phl p 1, Phl p 4, Phl p 12 and Phl p 2 and 6 (the last two were in the same band), respectively. No significant differences in band intensity with pH change were observed.

[0109] summary In most basic treatments (26 out of 28), the protein content was higher than in the native and untreated samples, confirming that the basic treatment contributed to the results.

[0110] Regarding the major allergen content, Phl p 5 levels were higher in the pH 8 treatment. For ELISA competition (REINA), there was no clear trend depending on the pH or base treatment used, although treatment with ammonium hydroxide showed high efficacy.

[0111] For ELISA inhibition (IgE), the highest values ​​(μg of 50% inhibition) corresponded to pH 10 and 11. The protein and allergen profiles were not significantly affected by the different pH treatments nor by the different bases.

[0112] general conclusion In general, base treatment gave good results in terms of protein concentration and major allergen content. Protein and major allergen profiles in SDS-PAGE were not affected by the base treatment.

[0113] Example 2: Olive (Olea europaea) The decolorized olive extract was obtained according to method steps AC. Protein content The highest protein content was obtained after treatment with methylamine pH 9 (862 μg protein / mg lyophilized extract), whereas the lowest content corresponded to treatment with sodium hydroxide pH 10 (441 μg protein / mg lyophilized extract). The average value was 696 μg protein / mg lyophilized extract (Table 4, Figures 11 and 12).

[0114] ELISA Competitive (IgE) - Biological Potency The median value was 302 HEPL / mg lyophilized extract. The highest value corresponded to the sample treated with sodium hydroxide pH 8 (582 HEPL / mg) and the lowest value was treated with methylamine pH 7 (128 HEPL / mg) (Table 4).

[0115] The highest biological efficacy was observed in the treatment at pH 8 (347 HEPL / mg) and the lowest at pH 9 (236 HEPL / mg) (Table 5, Figure 13). ELISA Inhibition (IgE) The amount of lyophilized extract required to reach 50% inhibition is inversely proportional to the potency of the extract. The number of micrograms of lyophilized extract required to reach 50% inhibition did not correlate significantly with the HEPL / mg value (Spearman Rank Order Correlation). The lowest value corresponded to urea at pH 9 (0.043 μg) and the highest value was lithium hydroxide pH 10 (0.132 μg) (Table 4). The average value for the decolorized samples was 0.088 μg.

[0116] The values ​​obtained at different pH levels were very similar (Table 5, Figure 15). Larger differences were obtained with the different bases: the highest values ​​were obtained with lithium hydroxide (mean value 0.108 μg) and the lowest with urea (0.057 μg) (Table 4, Figure 16).

[0117] Individual results table

[0118] [Table 4]

[0119] Summary of results analyzed by group

[0120] [Table 5]

[0121] [Table 6]

[0122] For native and untreated samples, standard deviations are not shown since only one sample was obtained. Immunoblotting and SDS-PAGE SDS and Western blots were performed on all destained samples and compared to the native extract.

[0123] All electrophoresis was performed on 15% T acrylamide gels under reducing conditions. The same amount of lyophilized extract (25 μg) was loaded in all lanes. Gels were stained with Coomassie R-250. Additionally, Western blot membranes were incubated with a serum pool (1 / 5 dilution) of patients expressing IgE against olive (O. europaea) (measured using ELISA). Afterwards, the membranes were incubated with α-IgE-PO and developed using chemiluminescence.

[0124] The most intense bands in the SDS-MS of the natural extract were observed at 20 and 18 kDa (in order of intensity, Figure 22). Both bands were identified by IHRP as Ole e 1, the major olive allergen. There were also bands at 10.5, 42, 48, 73 and 89 kDa. Other allergens reported to IUIS are Ole e 2 (profilin, 15 kDa), Ole e 3 (porcarin, 9 kDa), Ole e 4 (32 kDa), Ole e 5 (16 kDa), Ole e 6 (10 kDa), Ole e 7 (nsLTP, 9-10 kDa), Ole e 8 (21 kDa), Ole e 9 (46 kDa), Ole e 10 (11 kDa) and Ole e 11 (39.4 kDa).

[0125] The most important difference observed in the SDS decolorized samples was the decrease in the high molecular weight bands as the pH became more basic, especially at pH 11. However, in the case of treatment with urea, it was observed at pH 9.

[0126] Additionally, Western blots were performed (Figure 18). The most intense bands in the native extract corresponded to 19 and 17 kDa (Ole e 1). Other observed bands were 13, 34, 38, 48 and 74 kDa. Treatment with base did not show any clear difference. However, the 34 and 13 kDa bands were lost at high pH (pH 9 with urea, pH 10 and 11 with other bases).

[0127] Thin-layer chromatography Thin layer chromatography was performed on all samples and the results were compared to native. Reference standards (of plant origin) were also used as technical controls.

[0128] The results are shown in Figure 19. Up to five different flavonoids were observable in the olive samples. The intensity of the signal was higher in the native extract than in the decolorized sample. summary Protein content and ELISA Competitive (REINA) No significant differences were observed between groups.

[0129] ELISA Inhibition (IgE) No differences were observed between pHs, but between bases, with the lowest potencies (requiring more μg to reach 50% inhibition) corresponding to the sodium hydroxide and lithium hydroxide treated samples.

[0130] Protein and allergen profiles generally showed weaker high molecular weight bands as the pH increased (pH 9–10), especially when treated with urea. Thin-layer chromatography showed a decrease in the amount of dye during basic treatment.

[0131] general conclusion 1. In general, treatment of olive extract with base gave better results in terms of protein content compared to the native form.

[0132] 2. There was a loss of high molecular weight proteins (and allergens) with high pH treatment, which means an enrichment of major allergens (with low molecular weight). Example 3: Dermatophagoides pteronyssinus The decolorized Dermatophagoides pteronyssinus extract was obtained according to method steps A-C.

[0133] Protein content The highest protein content was obtained after treatment with ammonium hydroxide at pH 7 (710 μg protein / mg lyophilized extract), while the lowest content was obtained with CH at pH 8. 3 NH 2The average value for the decolorized samples was 5985 μg protein / mg freeze-dried extract (Table 7).

[0134] Protein contents were higher than those of the native extract in all treatments. In relation to the use of specific bases, the highest protein content values ​​were obtained with ammonium hydroxide, while NaOH treatment showed the lowest concentrations (Table 9, Figure 21).

[0135] Major Allergen Content The highest level of Der p 1 corresponded to the native extract (20.3 μg Der p 1 / mg lyophilized extract), followed by pH 7 and 9 (averages of 17.6 and 17.1 μg Der p 1 / mg lyophilized extract, respectively). The average value of decolorization (of the samples) was 16.1 μg Der p 1 / mg lyophilized extract (Figure 22, Tables 7 and 8).

[0136] Concerning treatment with the different bases, the samples treated with ammonium hydroxide and methylamine had the highest Der p 1 levels (average 18.6 and 18.4 μg Der p 1 / mg lyophilized extract, respectively) (FIG. 23, Table 9).

[0137] ELISA competition (IgE) The highest biological potency corresponded to samples treated with ammonium hydroxide at pH 7 (707 HEPL / mg lyophilized extract). The lowest values ​​corresponded to treatment with NaOH (185.5 and 168.3 HEPL / mg lyophilized extract at pH 10 and 11, respectively) (Table 7). The median value for the decolorized samples was 356 HEPL / mg lyophilized extract (Figures 29 and 30).

[0138] ELISA Inhibition (IgE) The number of micrograms of lyophilization required to reach 50% inhibition was inversely proportional to the HEPL / mg value.

[0139] The lowest 50% inhibition value corresponded to methylammonium pH 9, followed by the native extract (0.024 and 0.030 μg, respectively, Table 7). No clear differences were observed between the pH groups (Figure 26, Table 8). With regard to the bases used, the highest 50% inhibition value corresponded to treatment with LiOH (mean 0.043 μg). The lowest values ​​were observed for the crude extract, treatment with ammonium hydroxide, and the pH 6 sample (no treatment) (0.030, 0.39, and mean 0.04, respectively) (Figure 27, Table 9).

[0140] Individual results table

[0141] [Table 7]

[0142] Summary of results analyzed by group

[0143] [Table 8]

[0144] [Table 9]

[0145] For native and untreated samples, standard deviations are not shown since only one sample was obtained. Immunoblotting and SDS-PAGE SDS and Western blots were performed on all destained samples and compared to the native extract.

[0146] All electrophoresis was performed on 15% T acrylamide gels under reducing conditions. All lanes were loaded with the same μg of lyophilized extract (35 μg). Gels were stained with Coomassie R-250. Immunoblots were performed and proteins were transferred to a membrane, which was then incubated with a serum pool (1 / 10 dilution) of patients expressing IgE to D. pteronyssinus (measured using ELISA). The membrane was then incubated with α-IgE-PO and developed using chemiluminescence.

[0147] The most intense bands in the SDS of the native extract were observed at 31, 28 and 15 kDa (Figure 35). There were no significant differences in the protein profile in the SDS of the decolorized samples.

[0148] Note: Some bands were sequenced with IHRP from Dermatophagoides pteronyssinus. Der p 1 and Der p 3 were identified at 31 kDa bands, Der p 10 and Der p 8 were identified at 28 kDa bands, and Der p 2 was identified at 15 kDa using monoclonal antibody α-Der p 2.

[0149] Additionally, Western blots were performed (Figure 29). The most intense bands in the native extract corresponded to 15, 28, 37, 46 and 60 kDa. The 46 kDa band was weaker in the decolorized extract. In some cases the 60 kDa band disappeared (basic treatment), but the 80 kDa band appeared more intense compared to the native extract.

[0150] summary Protein content was not affected by the treatment (no significant difference between groups). The decolorized samples showed a higher protein content than the native extract; however, this difference was not significant (only one native sample). Even the pH 6 sample (untreated) showed a higher protein content than the native extract, with similar values ​​to the treated samples. Therefore, the increase in protein content compared to the native extract must be due to the more extensive purification of these samples (dialyzed more than five times).

[0151] Major Allergen Content Regarding Der p 1 and Der p 2 levels were affected by the use of urea but not by pH changes. ELISA competition (REINA) and inhibition With respect to the urea treated samples, the worse results (lower HEPL / mg and higher μg of 50% inhibition) corresponded to the urea treated samples.

[0152] Protein Profile showed no significant differences between the bleaching treatments, Allergen Profile The only common difference is urea treatment, which attenuates band intensity. Destaining Western blots with basic pH reduces recognition of bands at higher molecular weights.

[0153] general conclusion 1. Protein content is higher in the “bleached” extract than in the natural extract. 2. High pH treatment reduced the recognition of high molecular weight proteins in immunoblots.

Claims

1. A method for producing a decolorized allergen extract, comprising the steps of: a) basifying the natural allergen extract to a pH of 7-11 for 1 minute to 24 hours; and then b) subjecting the extract to a first molecular fraction removal step to remove molecules having a molecular size of less than 3.5 kDa; and c) Adjust the pH to neutral to produce a decolorized allergen extract. This includes The method according to claim 1, wherein the natural allergen extract is derived from a source material selected from the group consisting of Phleum pratense, Olea europaea, and Dermatophagoides pteronyssinus.

2. Further comprising polymerization, the polymerization comprising: d) contacting the decolorized allergen extract with an aldehyde; and then e) subjecting the extract to a second molecular fraction removal step to remove molecules having a molecular size of less than 100 kDa; and then f) carrying out step e) at 3-15°C, preferably 3-5°C, until the allergen extract has a conductivity of less than 210 μS / cm measured at room temperature and / or is free of aldehydes as measured by UV or visible scanning to obtain a decolorized polymerized allergen extract. The method of claim 1 , comprising:

3. 3. The method of claim 1, wherein the natural allergen extract is basified to a pH of at least 7.5, 8.0, 8.5 or 9.0, and the natural allergen extract is basified to a pH of no more than 11, 10.5, 10.0, 9.5, or 9.

0.

4. The method according to any one of claims 1 to 3, wherein step a) is carried out for 15 minutes.

5. The method of any one of claims 1 to 4, wherein the first and second small molecule removal steps b) and e) each independently comprise an ultrafiltration step, a diafiltration step, a dialysis step, or a filtration step.

6. The method according to any one of claims 1 to 5, wherein the pH in step c) is adjusted to between pH 7.0 and pH 7.5, for example between pH 7.3 and pH 7.4.

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