METHODS OF PURIFYING AN ALLERGEN EXTRACT

Abstract

The invention relates to processes for producing semi-purified and purified allergen extracts and pharmaceutical compositions and vaccines for use in the diagnosis and treatment of allergy. In one aspect of the invention, a process for producing a depigmented allergen extract is provided, the process comprising: a) basifying a native allergen extract; and b) removing molecules having a molecular size of less than 3.5 kDa; and c) adjusting the pH to neutrality; thereby to produce a depigmented allergen extract.

Claims

1. A process for producing a depigmented allergen extract, the process comprising: a) basifying a native allergen extract to pH 7 to 11 for 1 minute to 24 hours; 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) adjusting the pH to neutrality to produce a depigmented allergen extract.

2. The process according to claim 1, further comprising polymerisation, wherein the polymerisation comprises: d) contacting a depigmented allergen extract with an aldehyde; then e) subjecting the extract to a second molecular fraction removal step to remove molecules having a molecular size of less than 100 kDa; then f) carrying out step e) at 3-15 degrees centigrade until the allergen extract has a conductivity of below 210 pS/cm, measured at room temperature, and/or is absent of aldehyde as determined by UV or visible scanning, to obtain a depigmented polymerised allergen extract.

3. The process according to claim 1, wherein the native allergen extract is basified to a pH of at least 7.5 and the native allergen extract is basified to a pH of no more than 11.

4. The process according to claim 1, wherein step a) is carried out for about 15 minutes.

5. The process according to claim 2, wherein the first and second low molecular fraction removal steps b) and e) each independently comprise an ultrafiltration step, a diafiltration step, a dialysis step, or filtration.

6. The process according to claim 1, wherein the pH in step c) is adjusted to between pH 7.0 and pH 7.5.

7. A depigmented allergen extract obtainable according to the process of claim 1.

8. A depigmented polymerised allergen extract obtained or obtainable according to the process of claim 2.

9. The allergen extract according to claim 7, wherein the allergen extract is derived from a source material selected from food allergens, air-borne allergens, epithelial allergens or insect allergens.

10. The allergen extract according to claim 7, wherein the allergen extract is derived from a source material selected from Arachis hypogaea, Alnus glutinosa, Betula alba, Corylus avellana, Cupressus arizonica, Olea europaea, Platanus sp, Cynodon dactylon, Dactylis glomerata, Festuca elatior, Holcus lanatus, Lolium perenne, Phleum pratense, Phragmites communis, Poa pratensis, Ambrosia elatior, Artemisia vulgaris, Chenopodium album, Parietaria judaica, Plantago lanceolata, Salsola kali, Avena sativa, Hordeum vulgare, Secale cereale, Triticum aestivum, Zea mays, Acarus siro, Blomia tropicalis, Dermatophagoides farinae, Dermatophagoides microceras, Dermatophagoides pteronyssinus, Euroglyphus maynei, Lepidoglyphus destructor, Tyrophagus putrescentiae, Glycophagus domesticus, Chortoglyphus arcuatus, Alternaria alternata, Cladosporium herbarum, or Aspergillus fumigatus.

11. The allergen extract according to claim 7, wherein the source material is selected from peanuts, tree pollen, weed pollen, grass pollen, cereal pollen, fungi, moulds, mites, human hair, human dander, cat dander, cat hair, dog dander, dog hair, horse dander, horse hair, rabbit dander, rabbit hair, rat dander, rat hair, mouse dander, mouse hair, guinea pig dander, guinea pig hair, feathers, cockroaches, ants, fleas, bee venom, wasp venom, or arthropod allergens.

12. A method of treating an allergy of a subject comprising administering to the subject the allergen extract as defined in claim 7.

13. The method according to claim 12, wherein the allergy is a pollen allergy.

14. A pharmaceutical composition comprising an allergen extract according to claim 7.

15. A vaccine comprising an allergen extract as defined in claim 7.

16. The process according to claim 2, wherein step e) is carried out at a temperature of 3-5 degrees centigrade.

17. The process according to claim 3, wherein the native allergen extract is basified to a pH of at least 8.0.

18. The process according to claim 3, wherein the native allergen extract is basified to a pH of no more than 10.5.

19. The process according to claim 6, wherein the pH in step c) is adjusted to between pH 7.3 and pH 7.4.

20. The allergen extract according to claim 8, wherein the allergen extract is derived from a source material selected from Arachis hypogaea, Alnus glutinosa, Betula alba, Corylus avellana, Cupressus arizonica, Olea europaea, Platanus sp, Cynodon dactylon, Dactylis glomerata, Festuca elation, Holcus lanatus, Lolium perenne, Phleum pratense, Phragmites communis, Poa pratensis, Ambrosia elatior, Artemisia vulgaris, Chenopodium album, Parietaria judaica, Plantago lanceolata, Salsola kali, Avena sativa, Hordeum vulgare, Secale cereale, Triticum aestivum, Zea mays, Acarus siro, Blomia tropicalis, Dermatophagoides farinae, Dermatophagoides microceras, Dermatophagoides pteronyssinus, Euroglyphus maynei, Lepidoglyphus destructor, Tyrophagus putrescentiae, Glycophagus domesticus, Chortoglyphus arcuatus, Alternaria alternata, Cladosporium herbarum, Aspergillus fumigatus, tree pollen, weed pollen, grass pollen, cereal pollen, fungi, moulds, human hair, human dander, cat dander, cat hair, dog dander, dog hair, horse dander, horse hair, rabbit dander, rabbit hair, rat dander, rat hair, mouse dander, mouse hair, guinea pig dander, guinea pig hair, feathers, cockroaches, ants, fleas, bee venom, wasp venom, or arthropod allergens.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0119] FIG. 1 shows protein content of P. pratense extracts (determined using Lowry-Biuret method) of lyophilized samples obtained after different pH treatments. Error bars refer to the standard deviation of different samples' mean value.

[0120] FIG. 2 shows protein content of P. pratense extracts (determined using Lowry-Biuret method) of lyophilized samples obtained after treatment with different bases. Error bars refer to the standard deviation of different samples' mean value.

[0121] FIG. 3 shows Phl p 5 (Phleum major allergen) content of lyophilized samples obtained after different pH treatments. Error bars refer to the standard deviation of different samples' mean value.

[0122] FIG. 4 shows Phl p 5 (Phleum major allergen) content of lyophilized samples obtained after treatment with different bases. Error bars refer to the standard deviation of different samples' mean value.

[0123] FIG. 5 shows biological potency (ELISA competition) of P. pratense extracts of lyophilized samples obtained after different pH treatments. Error bars refer to the standard deviation of different samples' mean value.

[0124] FIG. 6 shows biological potency (ELISA competition) of P. pratense extracts of lyophilized samples obtained after treatment with different bases. Error bars refer to the standard deviation of different samples' mean value.

[0125] FIG. 7 shows μg necessary to obtain 50% inhibition of IgE binding to native extract of P. pratense extracts of lyophilized samples obtained after different pH treatments. Error bars refer to the standard deviation of different samples' mean value.

[0126] FIG. 8 shows μg necessary to obtain 50% inhibition of IgE binding to native extract of P. pratense extracts of lyophilized samples obtained after treatment with different bases. Error bars refer to the standard deviation of different samples' mean value.

[0127] FIG. 9 shows SDS of P. pratense extracts treated with different bases.

[0128] FIG. 10 shows western-blot of P. pratense extracts treated with different bases.

[0129] FIG. 11 shows protein content of O. europaea extracts determined using Lowry-Biuret method of lyophilized samples obtained after treatment with different pH. Error bars refer to standard deviation of different samples mean value.

[0130] FIG. 12 shows protein content of O. europaea extracts determined using Lowry-Biuret method of lyophilized samples obtained after treatment with different bases. Error bars refer to the standard deviation of different samples mean value.

[0131] FIG. 13 shows biological potency of O. europaea extracts determined using ELISA competition method of lyophilized samples obtained after treatment with different pH. Error bars refer to the standard deviation of different samples mean value.

[0132] FIG. 14 shows biological potency of O. europaea extracts determined using ELISA competition method of lyophilized samples obtained after treatment with different bases. Error bars refer to the standard deviation of different samples mean value.

[0133] FIG. 15 shows μg necessary to obtain 50% inhibition of IgE binding to native extract of O. europaea of lyophilized samples obtained after treatment with different pH. Error bars refer to the standard deviation of different samples mean value.

[0134] FIG. 16 shows μg necessary to obtain 50% inhibition of IgE binding to native extract of O. europaea of lyophilized samples obtained after treatment with different bases. Error bars refer to the standard deviation of different samples mean value.

[0135] FIG. 17 shows SDS of O. europaea extracts treated with different bases.

[0136] FIG. 18 shows western-blot of O. europaea extracts treated with different bases.

[0137] FIG. 19 shows thin layer chromatography for O. europaea. A, samples treated with sodium and lithium hydroxide; B, treatments with ammonium hydroxide, sodium hydroxide and urea; C, treatments with methylamine. All assays were compared with native extract. Standards are: 1, chlorogenic acid; 2, quercetin; 3, rutin trihydrate; 4, isoquercitrin; 5, quercitrin; 6, kaempferol 3-glucoside; 7, apigenin 7-glucoside.

[0138] FIG. 20 shows protein content of D. pteronyssinus extracts determined using Lowry-Biuret method of lyophilized samples obtained after treatment with different pH. Error bars refer to the standard deviation of different samples mean value.

[0139] FIG. 21 shows protein content of D. pteronyssinus extracts determined using Lowry-Biuret method of lyophilized samples obtained after treatment with different bases. Error bars refer to the standard deviation of different samples mean value.

[0140] FIG. 22 shows major allergen content of D. pteronyssinus extracts determined using specific ELISA sandwich kit (Indoor) for Der p 1 and Der p 2 of lyophilized samples obtained after treatment with different pH. Error bars refer to the standard deviation of different samples mean value.

[0141] FIG. 23 shows major allergen content of D. pteronyssinus extracts determined using specific ELISA sandwich kit (Indoor) for Der p 1 and Der p 2 of lyophilized samples obtained after treatment with different acid or base. Error bars refer to the standard deviation of different samples mean value.

[0142] FIG. 24 shows biological potency of D. pteronyssinus extracts determined using ELISA competition method of lyophilized samples obtained after treatment with different pH. Error bars refer to the standard deviation of different samples mean value.

[0143] FIG. 25 shows biological potency of D. pteronyssinus extracts determined using ELISA competition method of lyophilized samples obtained after treatment with different pH. Error bars refer to the standard deviation of different samples mean value.

[0144] FIG. 26 shows biological potency of D. pteronyssinus extracts determined using ELISA inhibition method of lyophilized samples obtained after treatment with different pH (μg necessary to obtain 50% inhibition of IgE binding to native extract). Error bars refer to the standard deviation of different samples mean value.

[0145] FIG. 27 shows biological potency of D. pteronyssinus extracts determined using ELISA inhibition method of lyophilized samples obtained after treatment with different bases (μg necessary to obtain 50% inhibition of IgE binding to native extract). Error bars refer to the standard deviation of different samples mean value.

[0146] FIG. 28 shows SDS of D. pteronyssinus extracts treated with different bases.

[0147] FIG. 29 shows western-blot of D. pteronyssinus extracts treated with different bases.

[0148] The present invention is illustrated by the following examples which detail processes for the preparation, purification and basification of extracts comprising allergens.

[0149] Methods A-C detail the processes used to make the allergen extracts.

[0150] Methods

[0151] A. Optional Defatting Process of Raw Allergen Material

[0152] Defatted extract was obtained. In general, homogenised material was defatted in acetone at 3-5° C., and filtered. This step was repeated until the acetone was transparent. The defatted material was recovered and dried at room temperature until all the acetone had been removed.

[0153] B. Preparation of Native Allergen Extract

[0154] Dried defatted material was weighed and extracted in 0.01 M PBS/0.15M NaCl in a proportion 1:10 for 4 hours at 3-5° C. under magnetic stirring. Afterwards, the solution was centrifuged for 30 minutes at 4° C. at 10.000 r.p.m. The resulting supernatant was collected and stored at 3-5° C. and the pellet was reconstituted in 0.01 M/NaCl 0.15M (1:10) and extracted overnight at 3-5° C. under magnetic stirring. The solution was centrifuged for 30 minutes at 3-5° C. at 10.000 r.p.m and the supernatant was collected and mixed with the previously obtained fraction. The combined extract was filtered using 0.45 μm pore size and extensively dialyzed in 3 kDa cut-off dialysis membranes until the conductivity was lower than 500 μS/cm. The extract was then filter sterilized using 0.22 μm pore size.

[0155] C. Preparation of Depigmented Allergen Extract

[0156] Native extract in aqueous solution and maintained at 3-5° C. was further treated using the following procedure. Under magnetic stirring, the pH of the solution was adjusted to pH 7-11 by addition of sodium hydroxide, lithium hydroxide, potassium hydroxide, urea, ammonium hydroxide or methylamine and maintained under these conditions for 15 minutes. Afterwards the extract was dialyzed in 3.5 kDa cut-off dialysis membranes with purified water for approximately 17 hours against 10 volumes of purified water at 3-5° C. Purified water was substituted 4 times during this period. After the base treatment, the extract was collected and the pH adjusted to 7.3-7.4 using 0.1M HCl. Finally the extract was sterile filtered until 0.22 μm, frozen and freeze-dried.

[0157] Immunological Characterisation

[0158] Protein Content

[0159] The protein content of native and depigmented extracts was measured by the Lowry Biuret method following the manufacturer's instructions.

[0160] Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

[0161] Protein profiles were identified by SDS-PAGE under reducing conditions (samples incubated with β-mercaptoethanol and heated for 10 minutes at 95° C.) in 2.67% C, 15% T acrylamide-acrylamide gels. Samples and Low Molecular Weight Standard (BioRad Laboratories, Hercules, Calif., USA) were run in the same gel. Gels were stained with 0.1% Coomassie Brilliant Blue R-250 (BioRad).

[0162] Immuno-Blot

[0163] Electrophoretically separated proteins (by SDS-PAGE) were transferred to a PVDF membrane (Trans-Blot® Turbo™ Transfer Pack, BioRad) and incubated overnight with sera from patients sensitized to each allergen (Plasmalab International, Everett, Wash., USA) diluted in 0.01M Phosphate Buffer Solution (PBS)-0.1% Tween. Plasmalab International operates in full compliance with Food and Drug Administration regulations. Specific IgE binding to the extract was detected with peroxidase-conjugated monoclonal antibodies, antihuman-IgE-PO (Ingenasa, Madrid, Spain), developed with luminol solutions (Western Immun-Star™ Western CTM Kit, Bio-Rad) and detected by chemiluminescence (ChemiDoc XRS, Bio-Rad).

[0164] Major Allergen Quantification

[0165] Major allergens were quantified using ELISA sandwich method using enzyme-linked immunosorbent assay detection kits (Indoor Biotechnologies, VA, USA). Briefly, Nunc Maxisorp plates (Thermo Scientific, Waltham, Mass., USA) were coated with a specific monoclonal antibody diluted in carbonate/bicarbonate buffer (pH=9.6) and incubated over night at 4° C. Afterwards, plates were blocked with BSA 1% in PBS 0.01 M-Tween 0.05%. Then, samples and standard were added in serial one half dilutions with BSA 1% in PBS 0.01 M-Tween 0.05%. Specific secondary monoclonal antibody (biotinylated) was added and streptavidin was finally used. Reaction with development solution (chromogen) was measured at OD 450 nm after stopping with sulfuric acid. Standard curve was obtained using a 4-parameters logistic fit by the least-squares method, where samples concentrations were interpolated to obtain the results.

[0166] ELISA Competition (IgE)

[0167] Native and depigmented extracts' capacity to inhibit IgE binding to each in-house reference preparation (IHRP) biologically standardized was compared. Nunc plates (Thermo Scientific) were coated with anti-IgE. A pool of serum from patients sensitized to the allergen was incubated in the plate. Dilutions of the sample and IHRP were incubated with the allergen labelled with peroxidase. The mixture was added to the coated plate and incubated. Afterwards, development solution (chromogen) was added, stopped with sulfuric acid and optical density (OD) measured at 450 nm.

[0168] ELISA Inhibition (IgE)

[0169] In vitro allergenic activity of the extracts (native and depigmented) was tested by means of ELISA inhibition, establishing the 50% inhibition point, using a native extract as reference. Plastic microtiter plates (Immulon 4HBX; Thermo Scientific) were coated with the native extract (10 μg of protein/ml) overnight. Serial 1:2 dilutions were made from the native and depigmented extracts in a Nunc F plate (Thermo Scientific). Each dilution was incubated with a serum pool for 2 hours at room temperature. Afterward, the dilutions of the extracts were transferred to the native coated plates and incubated for 2 hours. After washing, 100 μl of anti-human IgE peroxidase was added and let to stand for 30 minutes at room temperature. After washing, the plates were developed for 30 minutes (chromogen) and stopped with sulfuric acid (1 N).

[0170] Thin Layer Chromatography (TLC)

[0171] Plant flavonoids were used as positive controls. Controls and standards were applied over a TLC aluminium sheet silica gel 60F (Merck, Darmstadt, Germany). Ethyl-acetate:formic acid:acetic acid:water (100:11:11:27) was used as eluent, and developed using solution 1% methanolic diphenylboric acid-β-ethylamino ester followed by 5% ethanolic Poly(ethylene glycol)-4000.

EXAMPLES

Example 1: Phleum pratense

[0172] Depigmented Phleum pratense extract was obtained in accordance with method steps A to C.

[0173] Protein Content

[0174] Maximum protein content was obtained after treatment using methylamonium pH 8 (865 μg protein/mg lyophilized extract), and minimum content corresponds to potassium hydroxide pH 11 treatment (579 μg protein/mg lyophilized extract) (Table 1). Mean value across all pHs was 718 μg protein/mg lyophilized extract.

[0175] The highest protein content corresponded to pH 8 treatments (mean value 758 μg prot/mg lyoph), and the lowest to pH 9 (694 μg prot/mg lyoph) (Table 2) (FIG. 2). The protein content at all pHs between 7 to 11 was higher than native extract and the sample at pH 6, which was the original pH of the sample (sample treated the same as the depigmented samples, but without the basifying pH change).

[0176] Regarding different bases used, the highest protein content was achieved using lithium hydroxide (739 μg prot/mg lyoph), and the lowest with urea (668 μg prot/mg lyoph) (Table 3, FIG. 2).

[0177] Major Allergen Quantification

[0178] The lowest level corresponded to ammonium hydroxide treatment at pH 10 (13.0 μg Phl p 5/mg lyophilized 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 lyophilized extract, respectively). Mean depigmented value was 26.6 μg Phl p 5/mg lyophilized extract.

[0179] The highest major allergen content was obtained in treatments at pH 8 (31.5 μg Phl p 5/mg lyophilized extract) and the lowest at pH 10 (23.2 μg Phl p 5/mg lyophilized extract) (FIG. 3, Table 2). However, all treatments with bases yielded higher major allergen content than native extract and sample at pH 6 (without treatment).

[0180] The lowest major allergen content was obtained in treatments using sodium hydroxide (24.1 μg Phl p 5/mg lyophilized extract), and the highest using potassium hydroxide (29.1 μg Phl p 5/mg lyophilized extract) (FIG. 4, Table 3).

[0181] ELISA Competition (IgE)—Biological Potency

[0182] The highest biological potency corresponded to samples treated with methyl ammonium pH 7 and 9 (3052 and 2909 HEPL/mg lyophilized extract, respectively) (FIGS. 5 and 6, Table 1). The lowest value corresponds to treatments with potassium hydroxide (mean value of 984 HEPL/mg lyophilized extract), similar to native extract (952 HEPL/mg) (Table 3). Differences could be detected between ammonium hydroxide and other groups (potassium hydroxide P=0.010, Tukey Test; urea P=0.005, sodium hydroxide P=0.012, and lithium hydroxide P=0.049, Mann-Whitney), except for methylamine. There were also differences between methylamine and potassium hydroxide. Mean value of depigmented samples was 1685 HEPL/mg lyophilized extract, higher than results obtained with native extract and sample at pH 6 (without treatment) (952 and 1262 HEPL/mg lyophilized extract, respectively).

[0183] ELISA Inhibition (IgE)

[0184] Micrograms of lyophilized μg necessary to reach 50% inhibition did not show correlation with HEPL/mg values (Pearson Product Moment Correlation, P>0.050).

[0185] 50% inhibition values at pH 8 were significantly higher than treatments at pH 11 and 10 (P=0.030 and P=0.017, respectively, Mann-Whitney Rank Sum Test) (FIG. 7, Table 2). The lowest value corresponded to potassium hydroxide at pH 11 (0.007 μg), followed by ammonium hydroxide at pH 11 (0.020 μg, respectively) (Table 1). Mean value was 0.102 μg, similar to native extract and sample at pH 6 (0.105 and 0.097 μg, respectively).

[0186] Regarding the base used, the highest 50% inhibition values corresponded to treatments with urea (mean of 0.195 μg) (FIG. 8, Table 3). The lowest values were observed in methylamine treatment (mean value of 0.066 μg). Differences were detected between urea treatments and methylamine, ammonium hydroxide, lithium hydroxide, potassium hydroxide and sodium hydroxide.

[0187] Table of Individual Results

TABLE-US-00001 TABLE NO 1 Individual data μg prot/ μg PhI p 5/ μg Samples mg lyoph. mg lyoph. HEPL/mg 50% inh. Native 603.0 19.6 952.4 0.105 W/O treat. pH6 584.0 21.6 1261.9 0.097 pH 7 NaOH 795.3 21.2 765.0 0.116 pH 8 NaOH 764.3 25.8 1134.0 0.117 pH 9 NaOH 587.0 19.0 1055.1 0.144 pH 10 NaOH 755.0 27.2 2149.1 0.101 pH 11 NaOH 726.3 27.3 1902.0 0.099 pH 7 LiOH 724.3 26.6 1988.0 0.089 pH 8 LiOH 773.0 40.1 2538.0 0.131 pH 9 LiOH 753.0 26.2 1407.9 0.141 pH 10 LiOH 749.5 23.7 1441.4 0.040 pH 11 LiOH 693.0 24.9 1164.0 0.054 pH 7 KOH 776.0 31.5 1771.4 0.110 pH 8 KOH 751.5 34.7 933.5 0.158 pH 9 KOH 797.5 33.7 1098.0 0.140 pH 10 KOH 711.0 27.3 642.8 0.085 pH 11 KOH 578.5 18.2 475.6 0.007 pH 7 Urea 706.5 23.7 1355.7 0.235 pH 8 urea 689.5 29.2 1744.0 0.168 pH 9 urea 608.0 26.0 1053.6 0.182 pH 7 NH4OH 645.0 26.4 1897.3 0.081 pH 8 NH4OH 706.0 28.0 2378.3 0.129 pH 9 NH4OH 702.5 16.5 2666.2 0.109 pH 10 NH4OH 681.0 13.0 2487.5 0.068 pH 11 NH4OH 794.0 38.0 2336.6 0.020 pH 7 CH3NH2 665.0 24.7 3052.1 0.076 pH 8 CH3NH2 864.7 31.2 1885.8 0.084 pH 9 CH3NH2 718.0 27.0 2909.2 0.068 pH 10 CH3NH2 636.0 24.8 736.7 0.048 pH 11 CH3NH2 746.5 29.2 2209.1 0.054

[0188] Summary of Results Analysed by Groups

TABLE-US-00002 TABLE NO 2 Summary of data. Mean values of treatments performed with each pH ± standard deviation μg prot/ μg PhI p 5/ μg mg lyoph. mg lyoph. HEPL/mg 50% inh. Native 603.0 19.6 952 0.105 6 (W/O treat.) 584.0 21.6 1262  0.097 7 718.7 ± 59.4 25.7 ± 3.5 1805 ± 759 0.118 ± 0.060 8 758.2 ± 61.8 31.5 ± 5.2 1769 ± 645 0.131 ± 0.030 9 694.3 ± 82.1 24.7 ± 6.2 1698 ± 858 0.131 ± 0.038 10 706.5 ± 49.6 23.2 ± 5.9 1491 ± 824 0.068 ± 0.025 11 707.7 ± 80.9 27.5 ± 7.2 1617 ± 784 0.047 ± 0.036

TABLE-US-00003 TABLE NO 3 Summary of data. Mean values of treatments performed with each base ± standard deviation μg prot/ μg PhI p 5/ μg mg lyoph. mg lyoph. HEPL/mg 50% inh. Native 603.0 19.6 952 0.105 6 (W/O treat.) 584.0 21.6 1262 0.097 NaOH 725.6 ± 81.3 24.1 ± 3.8 1401 ± 593 0.115 ± 0.018 LiOH 738.6 ± 30.8 28.3 ± 6.7 1708 ± 553 0.091 ± 0.045 KOH 722.9 ± 86.9 29.1 ± 6.7  984 ± 503 0.100 ± 0.059 Urea 668.0 ± 52.7 26.3 ± 2.8 1384 ± 346 0.195 ± 0.036 NH4OH 705.7 ± 55.0 24.4 ± 9.9 2353 ± 285 0.081 ± 0.042 CH3NH2 726.0 ± 88.8 27.4 ± 2.8 2159 ± 930 0.066 ± 0.015

[0189] Native and samples without treatment do not present standard deviation since only one sample was analysed.

[0190] SDS and Western Blot

[0191] SDS and western-blot were performed with all depigmented samples compared with native extract (Phleum).

[0192] All electrophoresis were performed under reducing conditions, in acrylamide gels at 15% T. All lanes were loaded with the same μg of lyophilized samples (25 μg). Gels were stained with Coomassie R-250. Membranes were incubated with a pool of sera of patients presenting IgE to P. pratense (determined using ELISA) diluted 1/5. Afterwards, membranes were incubated with α-IgE-PO and developed using chemiluminiscence. SDS results are showed in FIG. 9. Western blots are shown in FIG. 10.

[0193] The most intense bands for the native extract were observed at 11, 37 and 31 kDa (in intensity order). The most important difference observed in SDS of depigmented samples was the decrease in intensity of high molecular bands as the pH increased, although this effect only led to less intense bands, and no bands were completely removed.

[0194] Note: Some bands have been sequenced in P. pratense IHRP. Phl p 5 was identified in the 37 kDa band, Phl p 1 in the 31 kDa band, and Phl p 2 (or 3) and 6 was identified in 12 kDa band. These allergens have been reported in the IUIS at 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 described 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 (profiling), at 14 kDa.

[0195] In addition, western-blots were performed (FIG. 10). The most intense bands in native extract corresponded to 37, 31, 59, 15 and 12 kDa (in intensity order), 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 important differences in band intensity were observed with pH change.

SUMMARY

[0196] In most basic treatments (26 out of 28) protein content was higher than native and untreated samples, confirming that the basic treatment is responsible for the results.

[0197] Regarding major allergen content, Phl p 5 levels were higher in pH 8 treatments.

[0198] In relation to ELISA competition (REINA), there was not a clear tendency depending on the pH or base treatment used, although treatments with ammonium hydroxide presented higher potency.

[0199] In relation to ELISA inhibition (IgE), the highest values (μg of 50% inhibition) corresponded to pH 10 and 11.

[0200] Protein profiles and allergenic profiles were not significantly affected with different pH treatments nor with different bases.

General Conclusions

[0201] In general, treatment with bases yielded better results in terms of protein concentration and major allergen content. Protein and major allergen profiles in SDS PAGE were not affected by the basic treatment.

Example 2: Olea europea

[0202] Depigmented Olea europaea extract was obtained in accordance with method steps A to C.

[0203] Protein Content

[0204] Maximum protein content was obtained after treatment using methylamine pH 9 (862 μg protein/mg lyophilized extract), and minimum content corresponds to sodium hydroxide pH 10 treatment (441 μg protein/mg lyophilized extract). Mean value was 696 μg protein/mg lyophilized extract (Table 4, FIGS. 11 and 12).

[0205] ELISA Competition (IgE)—Biological Potency

[0206] Medium value was 302 HEPL/mg lyophilized extract. The highest value corresponded to sample treated with sodium hydroxide pH 8 (582 HEPL/mg) and the lowest was treated with methylamine pH 7 (128 HEPL/mg) (Table 4).

[0207] The highest biological potency was observed in treatments at pH 8 (347 HEPL/mg), and the lowest at pH 9 (236 HEPL/mg) (Table 5, FIG. 13).

[0208] ELISA Inihibition (IgE)

[0209] The amount of lyophilized extract necessary to reach 50% inhibition is inversely proportional to the potency of that extract. Micrograms of lyophilized necessary to reach 50% inhibition did not present significant correlation with HEPL/mg values (Spearman Rank Order Correlation). The lowest value corresponded to urea at pH 9 (0.043 μg), and the maximum was lithium hydroxide pH 10 (0.132 μg) (Table 4). Mean value of depigmented samples was 0.088 μg.

[0210] Values obtained at different pH were very similar (Table 5, FIG. 15). Greater differences were obtained using different bases. The highest value was obtained with lithium hydroxide (mean value 0.108 μg), and the lowest with urea (0.057 μg) (Table 4, FIG. 16).

[0211] Table of Individual Results

TABLE-US-00004 TABLE NO 4 Individual data μg prot/ μg Samples mg lyoph. HEPL/mg 50% inh. Native 603.0 294.6 0.079 W/O treat. pH6 750.0 380.1 0.059 pH 7 NaOH 735.5 467.8 0.083 pH 8 NaOH 644.5 582.0 0.105 pH 9 NaOH 790.5 157.0 0.105 pH 10 NaOH 440.5 219.3 0.112 pH 11 NaOH 694.0 233.7 0.106 pH 7 LiOH 687.5 208.8 0.120 pH 8 LiOH 631.5 270.3 0.073 pH 9 LiOH 522.5 245.0 0.117 pH 10 LiOH 670.5 368.5 0.132 pH 11 LiOH 660.0 529.1 0.101 pH 7 KOH 722.5 206.1 0.084 pH 8 KOH 701.5 248.6 0.074 pH 9 KOH 747.5 182.1 0.072 pH 10 KOH 681.5 206.7 0.072 pH 11 KOH 648.5 322.0 0.079 pH 7 Urea 712.0 469.1 0.076 pH 8 urea 617.0 461.0 0.052 pH 9 urea 828.0 153.8 0.043 pH 7 NH4OH 706.0 168.2 0.083 pH 8 NH4OH 856.5 288.7 0.097 pH 9 NH4OH 814.5 439.7 0.077 pH 10 NH4OH 713.5 351.8 0.101 pH 11 NH4OH 552.0 211.3 0.066 pH 7 CH3NH2 750.0 127.9 0.096 pH 8 CH3NH2 540.0 233.3 0.095 pH 9 CH3NH2 862.0 238.1 0.091 pH 10 CH3NH2 795.0 430.1 0.066 pH 11 CH3NH2 774.5 429.1 0.092

[0212] Summary of Results Analysed by Groups

TABLE-US-00005 TABLE NO 5 Summary of data. Mean values of treatments performed with each pH ± standard deviation μg prot/ μg mg lyoph. HEPL/mg 50% inh. Native 603.0 294.6 0.079 6 (W/O treat.) 750.0 380.1 0.059 7 718.9 ± 22.2  274.7 ± 153.0 0.090 ± 0.016 8 665.2 ± 107.2 347.3 ± 141.5 0.083 ± 0.020 9 760.8 ± 122.9 235.9 ± 107.2 0.084 ± 0.026 10 660.2 ± 132.1 315.3 ± 97.9  0.096 ± 0.027 11 665.8 ± 80.5  345.0 ± 133.9 0.089 ± 0.016

TABLE-US-00006 TABLE NO 6 Summary of data. Mean values of treatments performed with each base ± standard deviation. μg prot/ μg mg lyoph. HEPL/mg 50% inh. Native 603.0 294.6 0.079 6 (W/O treat.) 750.0 380.1 0.059 NaOH 661.0 ± 134.4 331.9 ± 183.0 0.102 ± 0.011 LiOH 634.4 ± 65.8  324.3 ± 128.9 0.108 ± 0.023 KOH 700.3 ± 37.9  233.1 ± 55.1  0.076 ± 0.005 Urea 719.0 ± 105.7 361.3 ± 179.7 0.057 ± 0.017 NH4OH 728.5 ± 118.0 291.9 ± 108.7 0.085 ± 0.014 CH3NH2 744.3 ± 121.6 291.7 ± 133.4 0.088 ± 0.012

[0213] Native and samples without treatment do not present standard deviation since only one sample was obtained.

[0214] Immunoblot and SDS-PAGE

[0215] SDS and western-blot were performed with all depigmented samples compared with native extract.

[0216] All electrophoresis were performed under reducing conditions, in acrylamide gels at 15% T. All lanes were loaded with the same quantity of lyophilized extract (25 μg). Gels were stained with Coomassie R-250. In addition, western-blot membranes were incubated with a pool of sera of patients presenting IgE to O. europaea (determined using ELISA) diluted 1/5. Afterwards, membranes were incubated with α-IgE-PO and developed using chemiluminiscence.

[0217] Most intense bands in SDS of native extract were observed at 20 and 18 kDa (in intensity order, FIG. 22). Both bands have been identified in the IHRP as Ole e 1, major allergen of Olea. There were also bands at 10.5, 42, 48, 73 and 89 kDa. Other allergens reported in the IUIS are Ole e 2 (profilin, 15 kDa), Ole e 3 (polcalcin, 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).

[0218] The most important difference observed in SDS of depigmented samples was the decrease in high molecular bands as the pH is more basic, especially at pH 11. However, in the case of treatments with urea, it was observed at pH 9.

[0219] In addition, western-blots were performed (FIG. 18). The most intense bands in native extract corresponded to 19 and 17 kDa (Ole e 1). Other observed bands were at 13, 34, 38, 48 and 74 kDa. There were not clear differences in treatments with bases. However, bands at 34 and 13 kDa were lost at high pH (pH 9 with urea, pH 10 and 11 with other bases).

[0220] Thin Layer Chromatography

[0221] Thin layer chromatography was performed with all the samples and results were compared to the native. Reference standards (vegetal origin) were also used as technique control.

[0222] Results are shown in FIG. 19. Up to five different flavonoids could be observed in Olea samples. Intensity of signals was higher in native extract than in depigmented samples.

SUMMARY

[0223] Protein content and ELISA competition (REINA): no significant differences were observed between groups.

[0224] In relation to ELISA inhibition (IgE), no differences were observed between pHs, but between bases. The lowest potency (more μg needed to reach 50% inhibition) corresponded to sodium hydroxide and lithium hydroxide treated samples.

[0225] Protein profiles and allergenic profiles presented, in general, weaker high molecular bands as the pH increased (pH 9-10), especially when treated with urea.

[0226] Thin layer chromatography showed a decrease in the amount of pigments during basic treatment.

General Conclusions

[0227] 1. In general, treatment of O. europeae extracts with bases yielded better results in terms of protein content, compared to native. [0228] 2. There was a loss of high molecular weight proteins (and allergens) at high pH treatment, which implies an enrichment in major allergens (which have lower molecular weight).

Example 3: D. pteronyssinus

[0229] Depigmented D. pteronyssinus extract was obtained in accordance with method steps A to C.

[0230] Protein Content

[0231] Maximum protein content was obtained after treatment with ammonium hydroxide pH 7 (710 μg protein/mg lyophilized extract), and minimum content corresponds to CH.sub.3NH.sub.2 pH 8 treatment (561.5 μg protein/mg lyophilized extract) (Table 7). Mean value of depigmented samples is 5985 μg protein/mg lyophilized extract (Table 7).

[0232] Protein content was higher in all treatments than in native extract.

[0233] In relation to the use of a particular base, the highest protein content values were obtained with ammonium hydroxide, whilst NaOH treatments presented the lowest concentrations (Table 9, FIG. 21).

[0234] Major Allergen Content

[0235] The highest level of Der p 1 corresponded to native extract (20.3 μg Der p 1/mg lyophilized extract), followed by pH 7, and 9 (mean of 17.6 and 17.1 μg Der p 1/mg lyophilized extract, respectively). Mean depigmented value was 16.1 μg Der p 1/mg lyophilized extract (FIG. 22, Tables 7 and 8).

[0236] Regarding the treatment with different bases, Der p 1 levels of samples treated with ammonium hydroxide and methylamine (means of 18.6 and 18.4 μg Der p 1/mg lyophilized extract, respectively) are the highest (FIG. 23, Table 9).

[0237] ELISA Competition (IgE)

[0238] The highest biological potency corresponded to samples treated at pH 7 with ammonium hydroxide (707 HEPL/mg lyophilized extract). The lowest value corresponds to treatments with NaOH (185.5 and 168.3 HEPL/mg lyophilized extract at pH 10 and 11, respectively) (Table 7). Medium value of depigmented samples was 356 HEPL/mg lyophilized extract (FIGS. 29 and 30).

[0239] ELISA Inhibition (IgE)

[0240] Micrograms of lyophilized necessary to reach 50% inhibition were inversely proportional to HEPL/mg values.

[0241] The lowest 50% inhibition value corresponded to methylamonium pH 9, followed by native extract (0.024 and 0.030 μg, respectively, Table 7).

[0242] No clear differences were observed between pH groups (FIG. 26, Table 8). Regarding the base used, the highest 50% inhibition values corresponded to treatments with LiOH (mean of 0.043 μg). Lowest values were observed in native extract, treated with ammonium hydroxide and sample at pH 6 (without treatment) (0.030, 0.39 and mean of 0.04, respectively) (FIG. 27, Table 9).

[0243] Table of Individual Results

TABLE-US-00007 TABLE NO 7 Individual data. μg prot/ μg Der p 1/ μg Der p 2/ μg Samples mg lyoph. mg lyoph. mg lyoph. HEPL/mg 50% inh. Native 400.5 20.3 14.6 223.9 0.030 W/O treat. pH6 610.0 16.6 24.7 489.4 0.038 pH 7 NaOH 602.0 17.6 17.3 502.0 0.033 pH 8 NaOH 581.0 12.5 16.2 412.4 0.033 pH 9 NaOH 567.0 16.1 16.4 185.5 0.035 pH 10 NaOH 587.5 14.0 15.9 168.3 0.048 pH 11 NaOH 590.0 11.5 18.0 171.9 0.053 pH 7 LiOH 610.5 16.2 19.0 332.0 0.040 pH 8 LiOH 562.3 12.2 15.8 288.9 0.041 pH 9 LiOH 608.3 15.7 16.6 321.5 0.043 pH 10 LiOH 630.5 12.7 16.4 376.0 0.048 pH 11 LiOH 661.5 8.9 17.9 375.3 0.043 pH 7 KOH 655.0 16.1 19.6 404.2 0.031 pH 8 KOH 574.5 18.5 18.9 394.5 0.039 pH 9 KOH 611.5 14.7 19.4 360.6 0.046 pH 10 KOH 585.5 14.2 15.9 441.8 0.048 pH 11 KOH 581.0 11.0 20.0 331.0 0.047 pH 7 NH4OH 710.0 19.3 19.7 707.3 0.038 pH 8 NH4OH 612.5 19.5 16.6 468.9 0.037 pH 9 NH4OH 699.0 19.5 16.4 511.4 0.037 pH 10 NH4OH 701.0 19.2 16.4 671.1 0.034 pH 11 NH4OH 645.0 15.3 15.2 481.5 0.048 pH 7 CH3NH2 632.0 19.0 16.3 218.2 0.048 pH 8 CH3NH2 561.5 20.5 17.8 232.8 0.049 pH 9 CH3NH2 600.5 19.5 15.8 279.0 0.024 pH 10 CH3NH2 658.5 17.2 14.5 335.2 0.041 pH 11 CH3NH2 648.5 15.6 15.3 423.5 0.045

[0244] Summary of Results Analysed by Groups

TABLE-US-00008 TABLE NO 8 Summary of data. Mean values of treatments performed with each pH ± standard deviation. μg prot/ μg Der p 1/ μg Der p 2/ μg mg lyoph. mg lyoph. mg lyoph. HEPL/mg 50% inh. Native 400.5 20.26 14.63 223.9 0.030 6 (W/O treat.) 610.0 16.63 24.66 489.4 0.038 7 641.9 ± 44.6 17.6 ± 3.1 18.4 ± 1.5 432.8 ± 144.2 0.038 ± 0.006 8 578.4 ± 46.2 16.6 ± 3.3 17.1 ± 1.5 359.5 ± 145.4 0.040 ± 0.006 9 617.3 ± 45.7 17.1 ± 3.3 16.9 ± 1.5 331.6 ± 146.6 0.037 ± 0.007 10 632.6 ± 44.9 15.5 ± 3.3 15.8 ± 1.7 398.5 ± 140.8 0.044 ± 0.007 11 625.2 ± 44.4 12.5 ± 3.3 17.3 ± 1.7 356.6 ± 132.9 0.047 ± 0.007

TABLE-US-00009 TABLE NO 9 Summary of data. Mean values of treatments performed with each base ± standard deviation. μg prot/ μg Der p 1/ μg Der p 2/ μg mg lyoph. mg lyoph. mg lyoph. HEPL/mg 50% inh. Native 400.5 20.26 14.63 223.9 0.030 6 (W/O treat.) 610.0 16.63 24.66 489.4 0.038 NaOH 585.5 ± 12.8 14.35 ± 2.52 16.77 ± 0.85  288.0 ± 157.8 0.040 ± 0.009 LiOH 614.6 ± 36.2 13.16 ± 2.95 17.12 ± 1.31 338.7 ± 37.3 0.043 ± 0.003 KOH 601.5 ± 33.0 14.90 ± 2.74 18.76 ± 1.65 386.4 ± 42.4 0.042 ± 0.007 NH4OH 673.5 ± 42.6 18.55 ± 1.83 16.84 ± 1.67  568.1 ± 112.4 0.039 ± 0.005 CH3NH2 620.2 ± 39.5 18.35 ± 1.92 15.93 ± 1.22 297.7 ± 83.9 0.041 ± 0.010

[0245] Native and samples without treatment do not present standard deviation since only one sample was obtained.

[0246] Immunoblot and SDS-PAGE

[0247] SDS and western-blot were performed with all depigmented samples compared with native extract.

[0248] All electrophoresis were performed under reducing conditions, in acrylamide gels at 15% T. All lanes were loaded with the same μg of lyophilized (35 μg). Gels were stained with Coomassie R-250. Immunoblots were performed transferring proteins to membranes, which were incubated afterwards with a pool of sera of patients presenting IgE to D. pteronyssinus (determined using ELISA) diluted 1/10. Afterwards, membranes were incubated with α-IgE-PO and developed using chemiluminiscence.

[0249] The most intense bands in native extract SDS were observed at 31, 28 and 15 kDa (FIG. 35). There were no important differences in protein profile in SDS of depigmented samples.

[0250] Note: Some bands were sequenced in D. pteronyssinus IHRP. Der p 1 and Der p 3 were identified in 31 kDa band, and Der p 10 and Der p 8 were identified in 28 kDa band. Der p 2 was identified at 15 kDa using monoclonal antibody α-Der p 2.

[0251] In addition, western-blot was performed (FIG. 29). The most intense bands in native extract corresponded to 15, 28, 37, 46 and 60 kDa. 46 kDa band was weaker in depigmented extracts. In some cases, the band at 60 kDa disappeared (basic treatments), while band at 80 kDa appeared more intense compared to native extract.

SUMMARY

[0252] Protein content was not affected by the treatments (no significant differences between groups). Depigmented samples presented higher protein content than native extract. However, this difference was not significant (only one native sample). Even sample at pH 6 (without treatment) presented higher protein content than native extract, similar value to treated samples. So the increase in protein content compared to native extract must be due to the higher purification of these samples (they are dialyzed 5 times more).

[0253] Regarding major allergen content, Der p 1 and Der p 2 levels were affected by the use of urea, and not by the pH change.

[0254] In relation to ELISA competition (REINA) and inhibition, the worse results (lower HEPL/mg and higher μg of 50% inhibition) corresponded to urea treated samples.

[0255] Protein profiles did not show important differences between depigmentation treatments, while allergenic profiles did so. The only common differences refer to urea treatments, that reduce bands intensity. Western blot of depigmented with basic pH decrease band recognition in high molecular weight.

General Conclusions

[0256] 1. The protein content is higher in “depigmented” extracts than in native extracts. [0257] 2. High pH treatments decreased the recognition of high molecular weight proteins in an immunoblot.