Method for the production of hydrolyzed allergens

Abstract

A pharmaceutical composition of hydrolyzed allergens and its use for treating, curing or preventing allergic reactions. The hydrolyzed allergens are prepared by: a) extracting a source of allergens comprising allergenic proteins to form an extract, b) purifying the extract to remove non-protein components to form a purified extract, c) denaturing the purified extract with a first denaturing agent to form a purified denatured extract, d) refining the purified denatured extract to remove impurities to form a refined denatured extract, e) denaturing the refined denatured extract with a second denaturing agent to form denatured allergen mixture, and f) hydrolyzing the denatured allergen mixture to form the hydrolyzed allergens.

1 . A pharmaceutical composition comprising hydrolyzed allergens obtained by
 a) extracting a source of allergens comprising allergenic proteins to form an extract,   b) purifying the extract to remove non-protein components to form a purified extract,   c) denaturing the purified extract with a first denaturing agent at a pH of 7.0 to 11.0 to form a purified denatured extract,   d) refining the purified denatured extract to remove impurities to form a refined denatured extract,   e) denaturing the refined denatured extract with a second denaturing agent at a pH of 1.0 to 7.0 to form denatured allergen mixture,   f) hydrolyzing the denatured allergen mixture to form the hydrolyzed allergens, and   g) purifying the hydrolyzed allergens to remove peptides with molecular weights above 10,000 Da and below 1,000 Da, wherein 70% or more of the peptides are between 10,000 Da and 1,000 Dawherein the first denaturing agent is a mixture of a chaotropic agent and a reducing agent and wherein the second denaturing agent is a mixture of a chaotropic agent and a reducing agent.   
     
     
         2 . The pharmaceutical composition of  claim 1 , further additionally at least one substance selected from the group of nucleoside triphosphates, nucleoside diphosphates, nucleoside monophosphates, nucleic acids, peptide nucleic acids, nucleosides or analogs thereof, immunosuppressive cytokines, compounds inducing expression of immunoproteasomes, 1,25-dihydroxyvitamin D3 or analogs thereof, lipopolysaccharides, endotoxins, heat shock proteins, thioredoxin with either NADPH or NADP-thioredoxin reductase, reducing agent, dithiothreitol, adrenergic receptor agonists such as salbutanol, adrenergic receptor antagonists such as butoxamine, compounds that regulate the expression of the adhesion molecule ICAM-1, N-acetyl-L-cysteine, y-L-glutamyl-L-cysteinyl-glycine (reduced L-glutathione), alpha-2-macroglobulins, inducers for Foxp3 gene expression, flavonoids, isoflavonoids, pterocarpanoids, stilbenes such as resveratrol, tachykinin receptor antagonists, chymase inhibitors, vaccine adjuvant or immunomodulators like CpG, aluminum hydroxide, calcium phosphate, TLR-4 agonists (i.e. MPL) and TLR-9 agonists or tolerogenic adjuvant like zymosan, beta1,3-glucan, regulatory T-cell inducer, a muco-adhesive agent for attaching the particle to the intestinal mucosal lining such as a plant lectin, zinc, zinc salts, polysaccharides, vitamins and bacterial lysates or particles displaying surface linked antibodies. 
     
     
         3 . The pharmaceutical composition according to  claim 1 , for oral, subcutaneous, nasal, epicutaneous or intralymphatic administrations, for sublingual drug delivery, or for enteric drug delivery. 
     
     
         4 . The pharmaceutical composition of  claim 1 , wherein extracting is performed in a solution comprising no salt or a salt selected from carbonate, bicarbonate, phosphate, acetate, TRIS and HEPES, wherein extracting is performed with an extraction medium. 
     
     
         5 . The pharmaceutical composition of  claim 1 , wherein the purifying and/or refining comprises one or more of an ion exchange chromatography step, a gel filtration or size exclusion chromatography step, a hydrophobic interaction chromatography step, a pseudo affinity or affinity chromatography step. 
     
     
         6 . The pharmaceutical composition of  claim 1 , wherein denaturing is performed with a denaturing agent selected from the group of chaotropic agents, reducing agents and mixtures thereof. 
     
     
         7 . The pharmaceutical composition according to  claim 6 , wherein the first and/or second denaturing agent comprises urea at a concentration of more than 4 M, and guanidinium chloride at a concentration above 3 M. 
     
     
         8 . The pharmaceutical composition of  claim 1 , wherein hydrolyzing is performed with an enzyme. 
     
     
         9 . The pharmaceutical composition of  claim 1 , wherein the removal of the peptides is performed by size exclusion chromatography and/or by ultrafiltration, wherein the size exclusion chromatography is performed in the presence of chaotropic agents. 
     
     
         10 . The pharmaceutical composition of  claim 1 , wherein the sources of allergens are peanuts. 
     
     
         11 . The pharmaceutical composition of  claim 8 , wherein the hydrolyzing is performed in the presence of a chaotropic agent and a reducing reagent. 
     
     
         12 . The pharmaceutical composition of  claim 10 , wherein the source of allergens is a mixture of at least two species/subspecies/varieities/hybrids and/or transgenic peanuts. 
     
     
         13 . The pharmaceutical composition of  claim 12 , wherein the peanuts are selected from the  Arachis  genus. 
     
     
         14 . The pharmaceutical composition of  claim 13 , wherein the peanuts selected from the  Arachanis  genus are  hypogaea  or  fastigiata.    
     
     
         15 . The pharmaceutical composition of  claim 1 , wherein denaturing is performed with a denaturing agent selected from the group of urea, guanidinium chloride, dithiotreitol, thiglycerol, β-mercaptoethanol, tris (2-carboxyethyl) phosphine (TCEP), and mixtures thereof. 
     
     
         16 . The pharmaceutical composition of  claim 1 , wherein
 extracting is performed in a solution comprising phosphate,   purifying or refining includes a precipitation step,   the precipitation step is performed with a solution comprising trichloroacetic acid,   denaturing is performed with a combination of urea and dithiotreitol,   the hydrolyzing is performed with pepsin,   the removal is performed by size exclusion chromatography in the presence of chaotropic agents, and   the source of allergens are peanuts from  Arachis hypogaea.      
     
     
         17 . The pharmaceutical composition of  claim 1 , wherein the second denaturing agent is tris(2-carboxyethyl)-phosphine (TCEP). 
     
     
         18 . A method for the treatment, cure or prevention of allergic reactions comprising administering the pharmaceutical composition of  claim 1 .