CA2145169C - A process for the purification of aqueous extracts containing allergenically active proteins, extracts obtainable according to this process as well as their use - Google Patents

Abstract

The invention concerns the removal of various allergologically irrelevant low-molecular weight components from the usual aqueous extracts of allergenically active proteins of plant pollens. Described are the desorption and subsequent elimination, from traditionally prepared allergenic pollen protein extracts, of low-molecular weight pigment and other compounds which are nor-mally retained by strong electrostatic and/or hydrophobic forces. The preparation of such depigmented pollen proteins does not impair their allergenic potency or immunological specificity. The invention enables the production of fully active allergenic poll-en proteins devoid of adhering low-molecular weight substances interfering with their safety, diagnostic accuracy and clinical ef-ficacy. The purified pollen proteins represent improved starting materials for chemical derivatization, i.e., the preparation of at-tenuated vaccines for immunotherapy. The invention also provides suitable products for investigating the molecular structure of allergenic epitopes.

Description

~O 94/06821 1 ~ ~ ~ ~ ~ ~ ~ PCT/NL92/00160 A process for the purification of aa~Pn". extracts containine al~ergenica~iv active proteins extracts obtainable according to this vrocess as well as their use The present process relates to a process for the purification of aqueous extracts containing allergenically active proteins and further containing non-allergenic undesirable compounds.
Aqueous extracts of the pollen grains of grasses, weeds, trees and other plants have since the turn of this century found widespread application for the in vivo and in vitro diagnosis of hayfever ("pollinosis") in predisposed human, so-called "atopic"
patients. Since the first description by Noon and Freeman in 1911, such extracts have also been used for the treatment of this ailment by applying them in a regimen of infections for long-term "desensi-tization", "hyposensitization", or "immunotherapy". Based on the observation that the causative mayor allergenic components in extracts of pollen are proteins in the molecular weight range of 20 - '70 kD, whereas the constituents below the 10 kD molecular weight range are believed to be non-allergenic, it has become common practice in the manufacturing process of pollen extracts for diagnosis and immunotherapy to dialyse or ultrafilter the aqueous pollen extracts through membranes of 5-10 kD nominal cut-off in order to remove supposedly irrelevant components with a molecular size lower than 5-10 kD, thereby retaining the allergenic proteins in the molecular size range of 10-100 kD in order to improve the quality of the allergenic extract for clinical application.
A number of reports has nevertheless in the past appeared in the scientific literature relating to the possible allergenic properties of the low-molecular weight and dialysable constituents of aqueous pollen extracts, i.e. substances with an upper limit mol-ecular weight of about 10 kD ( Moore MB and Moore EE. J Am Chem Soc 1931; 53: 244; Unger L, Cromwell HW, Moore MB. J Allergy 1932; 3:
253).
These investigations indicated that the low-molecular consti-tuents of M < 5 kD from pollen extracts do indeed exhibit some resi-dual allergenic activity, although their potency on a weight basis is a factor of at least 1000 less than that of the non-dialysable WO 94/06821 ~ PCT/NL92/00~

2 glycoproteins of M > 5 kD. These results, together with the highly complex chemical composition of the dialysable fraction of pollen extracts provided little impetus for pursueing these studies. The state of the prior art therefore is that the low-molecular weight components of M < 5 kD in pollen extracts are irrelevant in terms of their allergological and immunological contribution.
However, the content and biological activity of the water--soluble flavonoid-glycosides present in nearly every pollen extract has neverthless remained ambiguous. It is usually tacitly assumed that such compounds, which may considerably influence cellular func-tions in man and animals after parenteral administration, are being removed during the dialysis process (Wiermann R, Wollenweber E, Rehse C, Z Naturforsch 1981; 36 c: 204). Nevertheless, spectroscopic examination of the dialysed conventional pollen extracts containing proteins of M > 10 kD for diagnosis and therapy shows that a very high proportion of (flavonoid-) pigments remains firmly adsorbed to the proteins (compare Figure 1).
The present invention provides a process for the purification of aqueous extracts containing allergenically active proteins and further containing non-allergenic undesirable compounds, which process yields highly purified extracts containing substantially allergenically active proteins, which extracts do not suffer from the (not always recognized) disadvantages of the conventional aqueous extracts containing allergenically active proteins.
In a first embodiment the present invention relates to a process for the purification of aqueous extracts containing aller-genically active proteins and further containing non-allergenic undesirable compounds, wherein said non-allergenic compounds adhering to said proteins are removed from said proteins by using means which disrupt electrostatic forces and/or hydrophobic forces being responsible for the adherence of said non-allergenic compounds to said proteins.
Specifically, the process according to the invention com-prises the following steps:
1) providing an aqueous extract containing allergenically active proteins) and further containing non-allergenic undesirable compounds as a starting material, ~WO 94/06821 ~ ~ ~ ~~ PCT/NL92/00160

3 2) subjecting said starting material to a treatment to remove said non-allergenic compounds, which adhere to said proteins, from said proteins in which means are used which disrupt electrostatic forces and/or hydrophobic forces being respon-sible for the adherence of said non-allergenic compounds to said proteins, to obtain an aqueous extract substantially free from said adhering undesirable compounds, 3) collecting the substantially pure extract.
Farther embodiments of the present invention will be apparent from herebelow.
According to the invention it was established that impurities such as flavonoids and/or glycosides, but also other compounds, exemplified in the below, are contained in the usual extracts, but not in a "free" form. The "adherence" of the impurities to the allergenically active proteins may be based on Van der Waals forces, ionic bonding, hydrophobic interaction or even chemical interaction (covalent forces).
The expression "means which disrupt electrostatic forces, hydrophobic or other physical forces" relates to various chemical and/or physical means which are effective for the removal of adsorbed or (firmly) adhered compounds (impurities) from the aller-genically active proteins.
In a preferred embodiment of the process of the invention the means for disrupting electrostatic or other physical forces are selected from the group of chemical means consisting of acid, and alkaline materials including anion- and cation-exchanging materials, salts and electric currents.
It will be apparent that the removal of adsorbed impurities may not only be performed by changing the electrical environment (electrical charges) in the extract used as starting material, but also by the use of forces which influence the dielectric constants of the proteins in question. Means which influence hydrophobic interactions and Van der Waals forces may equally be used.
In the process of the invention it is preferred to use chemical means, in particular acid, in an amount causing the exceeding of the iso-electric point of the allergenically active proteins aimed at. It is preferred to use an acid having a pH-value WO 94/06821 w PCT/NL92/00 N .
of less than 3, preferably a pH-value in the range of 1.5-2.5. It will be clear that these pH-values are in general below the iso-electric points of almost every protein.
In the case of the use of an alkali a pH-value of e.g. 9-11, i.e. above the usual electric points, may be used.
r This means that the acid and alkali concentrations may be in the order of 0.1 N to 0.01 N for (strong) acids and 0.01-0.001 N for (strong) alkali.
If, on the other hand, it is desirable to dissociate or desorb low-molecular compounds with salts at neutral pH in the range of e.g. pH 6-8, the absolute salt concentration or ionic strength is relevant. Preferably, monovalent salts are used, e.g. NaCl, KC1, KCNS, the corresponding bromides, or e.g. guanidine-HC1. It is preferred that the value of the ionic strengths is between 2-6 M, preferably 2-5 M. It should be noted that higher concentrations, e.g. of guanidine-HC1, tend to disrupt hydrogen bonds of the carrier protein as well as inter-chain linkages of composite proteins. How-ever, on the other hand such a disruption may not be disadvantageous as in many cases the allergenity or antigenicity may be retained. It is supposed that these immunological properties are maintained by virtue of epitopes on the secondary structure of the proteins, which are responsible for this specific activity.
In a further embodiment of the present invention the means for disrupting electrostatic forces and physical forces comprise electric currents in the form of electrophoresis. Normally, the electrophoresis is carried out with the aqueous extracts. Normally, electrophoresis should not be performed too long in order to avoid local heat exposure or (complete) denaturation bf the protein.
Furthermore, a reasonably high electrical potential across the applied electrodes should be used, e.g. in general between 10-2000 Volts DC, in particular between 200-1000 Volts DC. If the potential is too high, the protein carriers themselves may suffer some pertub-ation of their colloid-chemical zeta potential, thereby irreversibly causing unfolding of the tertiary or even secondary structure and a loss of the stabilizing water layer. Of course, the electrical cur-rent is dependent on the salt concentration in the solution, ..
normally a buffer solution.

WO 9,1!46821 i'CT/NL421p0160 Although aecordin8 tc the process of the iaveatzon narmahy non-~.Iergenfc aampou~nds having a t~plecular weight of ; less than 1.000 , preferably less rhea X040 are removed, it wi31 be apparent that with ~us1 good results also compounds having a lower molecular 5 weight, e.g. less than 1600. oay be removed i'ro~o the proteins.
However, preferably flarronoids and/or glycosides are removed.
Flavonoids ~ occurring in the present aqueous entreats a~'~ect srachidonic acid metabolism end interact as phenols or quinofd derivatives with m~crosrolecuies, causing for example esszyme i.rasctivation due to complex !'ormation with tann~.a-Zt~e polymers.
Strong interaction With proteins is in fact a general property of polyphenols (among them the flavonoids), their axidatfon prcducts~
mmad their polymers. As mentioned in the above the interaction of the impurities being present ia~ the extracts used as stertshg materials in the process of the irsventfon can be physical (Very der Wsals farces, ionic bonding, hydrophobic interactions) for chemical (covalent forces by interaction with oxidized (bi-)phrnols.
Flevoaoids and flavot~cidglyeoaides as individual molecules inhibit.
histamine release from mast cells end basophils arid modulate to a ?0 eansiderable degree the normal f'~xnct3.ons of pr~lymarphonuclear leuco-cytes and neutrophils. fihese biological nctiviti.es say have considerable impact on. the outcome of diagnostic skin arid inhalation tests with prior art preparations routinely performed in allergic patients. On the other head, ft hgs already been exta,~ively docu-mented in EP 0387952 that low-molecular weight (M < 5000) water-soluble and non-adsorbed flavonoid-glycosides do not contribute to the binding of IgE antibodies in the serum of specifically sensitized allergic patients. The removal of impurities such as flavonoids and their glycosides from the ~0 customary diagnostic and therapeutic pollen protein vaccines may therefore improve their usefulness and efficacy in clinical medicine.
A further argument for eliminating adsorbed low-molecular compounds from plant pollen proteins administered to man is that many pollen species, like the plants elaborating them, may contain low-mass (M < 1000) non-flavonoid organic compounds, potentially harmful to man, e.g. toxic alkaloids, benzochinones, terpenoids and WO 94/06821 ~ PCT/NL92/001~

their derivatives irritating to mucous membranes, and other aromatic structures. Like the flavonoids, such components may in some instances resist simple dialysis or ultrafiltration at neutral pH
through membranes of 10 kD nominal cut-off by remaining firmly adsorbed to proteins by physical forces.
The invention also relates to extracts obtainable according to the processes of the invention as described herein. Such purified , extracts ensure the safety of allergenic plant pollen extracts intended for the diagnosis and treatment of allergic diseases.
According to the present invention a broad variety of extracts of naturally occurring materials may be (further) purified.
Representative examples are:
Pollen Extracts The following tree pollens, grass pollens and weed pollens:
Tree pollens:
Acacia Longtfotta, Acacia baitegana, Ailanthus aZttssima, AZrms terut t f o t t a/i cnana, A Zrtus rubra, A Zeus a t rtuata, Prurats amygda Zus, Pbzus maZus (Matus pumtta), Prunus armeniaca, Thu,fa ortentatts, Fra xtrcus vetuttna, Fraxtnus ntgra, Fzaxtmcs pennsytvantca, Fzaxtnus ozegona, Potutus tremutotdes, Myzica gate, Fagus grandtfoZta, Betuta tenta, Betuta papyrtfera, BetuZa fonttnaZts, BetuZa atba, BetuZa verrucosa, BetuZa tutea, Carptmcs cazottneana, Catttstemon cttztracs, JugZans ctnerea, Ceratonta stttqua, Cedrus deodoza, Thu~a pttcata, Linocedrus decuzrens, Cryptomerta ~apontca, Chamaecypazts Zamsontana, Juntpezus vtrgtntana, Juntperus scoputozum, Tamaztx gaZ-Zica, Thu~a occZdentaZts, Prunus cerasus, Castanea dentata, Aescutus hippocastamrm, PopuZus trtchocazpa, Poputus deZtotdes, Poputus fzemonttt, Cupressus arizontca, Taxodtum distichum, Cupzessus sempezvtzens, Cupressus macrocarpa, Sambucus gtauca, Utmus crasst-fotta, Utmus parvtfotta, Utmus pumtta, UZmus fuZva, Eucalyptus gZobutus, Pseudotsuga menztestt, Abtes nobttts (procera), Abtes co~-cotoz, Ltqutdambar styzaciftua, CeZtts occtdentatts, CozgZus amertcana, Tsuga canadensts, Tsuga heterophytZa, Carya ovata, Carga tactniosa, Cazya tomentosa, Ostrya vtrgintana, Juntperus caZifor-ntca, Juntperus chtnensis, Jurctpezus monosperma, Juntperus 21 ~ ~~ 69 ~O 94/06821 ; : PCT/NL92/00160 i E.._f t y_ pinchotti, Jutntperus osteosperma (Juniperus utahensis), Juntperus occfdentaZis, Syrtnga vutgaris, Tita americana, Robins pseudoacacta, Acer macrophgttum, Acer saccharum, Acer rubrum, Acer saccharinum, Acer negundo, Metateuca teucadendron, Prospopts ~uttftora, PhtZadeZphus Zemtsti, Broussonetia papXtfera, ?focus rubra, Morus atba, Quercus gambetii, Quercus chrbsotepsts, Quercus vetuttna, Quercus maritandica, Quercus macrocarpa, Quercus keZZoggtt-catiforntca, Quercus dumosa, Quercus agrifoZia, Quercus engetmantt, Quercus garrbana, Quercus itex, Quercus ~istenzenit, Quercus stetZata, Quercus cobra, Quercus patustris, Quercus Zobata, Quercus vtrginiana, Quercus nigra, OZea europaea, Citrus stnensis, MacZura ptmifera, Phoenix dactyttfera, Chamaerops humutis, Phoenix canartensis, Cocos pZumosa, Prunus persica, Pyrus communts, Carya pecan, Schinus motte, Schinus terebinthtfotius, Casuartna equtsett-fotta, Ptnus nigra, Pinus canartensts, Pinus sabiniana, Pinus taeda, Pinus contorts, Ptnus radiata, Pitlus eduZis, Pinus resinosa, Pinus echtnata, Pinus vtrginiana, Pfracs pondersa, Ptnus strobus, Pinus monticota, Prunus domestics, Poputus batsamtfera, Poputus ntgra-tta-Zica, PoZuZus trichocarpa, PopuZus sibs, Sequofa sempervirens, EZaeagnus angustifotta, Picea rubens, Picea sttchensis, PZatanus occidentatis, PZatanus acerifotia, Ptatanus racemosa, Larix occiden-tatis, Tamarix gatttca, Ailanthus attissima, Jugtans rupestrts, Jugtans ntgra, Jugtans hindsti, JugZans catifornica, Jugtans regta, SaZtx Zastotepis, SaZix nigra, SaZtx discolor, Satix Zaevigata, Satix Zasiandra, Juniperus sabtnoides, Ptantago ZanceoZata, Fraxinus amertcana, Quercus aZba, Aces negundo, Atnus rhombifotta, UZmus americana;
Grass and iJeed Pollens:
Xordeum vuZgare, Agrostis tenuts, Poa annua, Poa compressa, Poa pratensis, Poa sandbergti, Bromus rigidus, Bromus carinatus, Bromus secaZinus, Bromus tnermts, Bromus moths, Agropyron sptcatum, PhaZaris canariensis, PhaZarts arundinacea, Festuca cobra, Boutetoua graciZis, Koeterta cristata, Eragrostfs vartabtZis, Avena sattva, Avena etatior (Arrhenatherum etatius), Agropyron repens, Agrostts atba, SecaZe cereate, Etymus triticotdes, Etymus cinereus, Lotium muttt,~torum, Etymus gtaucus, DistichZts stricta, Sorghum vutgare, tr ~ i~ '1 w WO 94/06821 ~ ' ' ~ PGT/NL92/00JI~

Sorghum vutgare var. Sudanese, Anthoxanthum odoratum, Hotcus Ianatus, Triticum aestivum, Agropbron smithif, Hedicago sativa, Aseter sinensis, BaZsamorhiza sagittata, Bassia hyssopifotia, Franseria bipinnatifida, HymenocZea satsota, Amaranthus patmeri, ricinus communis, THpha tatifotia, Trifottum pratense, MeZftotus w offtcinaZis, Trifotium repens (album), Xanthtum strumarium, Xanthium spinosum, Cosmos bipinnatus, Narcissus pseudonarcissus, DahZta pinnata x coccinea, Chrysanthemum Zeucanthemum, Taraxacum officinate, Rumex obtusifotius, Rumex cripus, Anthemix cotuZa, Epi-Zobitum angusti,~oZium, Gtadiotus Xhortutanus, Sarcobatus vermicutatus, Cannabis sativa, HumuZus Zuputus, Grayia spinsa, AtZenroZfea occidentaZis, Kochia scoparia, Litium ZongifZorum, Tagetes patuta, Iva xanthifoZia, Iva angustifotia, Zva ciZiata, Chenopodium ambrosiodes, Brassica nigra, Brassica campestris, Urtica diotca, Saticorrtia ambigua, Amaranthus retrofZexus, Amaranthus spinosus, Eschoschotzia caZiforntca, Iva axiZtaris, Chrysothamnzts nauseosus, Franseria deZtoides, Franseria ambrosiodes, Franseria dumosa, Franseria acanthicarpa, Ambrosia trifida, Ambrosia artemtsiifoZia (etatior), Dicoria canescens, Franseria tenui,~oZia, Ambrosia bidentata, Rosa muttifZora, Arthemisia caZifornica, Artemisia dracuncutus, Artemisia vutgaris heterophytta, Artemisi frigida, Artemisia pgcnocephata, Artemisia Iudovician, AtripZex rvrightii, AtripZex potycarpa, AtripZex serenana bracteosa, Atriptex tentiformis bremeri, Atriptex Zentiformis, Atriptex roses, AtripZex argentea expansa, Atriptex patuta hastata, AtripZex canescen, Cytisus scoparius, Suaeda catifornica, Carex barbara, AtripZex confertffotfa, Rumex acetosetZa, Antirrhinum ma,fus, Beta vutgaris, HeZianthus annuus, Acnida tamariscina, Eurotia tanata, Chenopodium botrbs, Artemtsia absinthium, Parietaria ~udatca, Parietaria officinaZis.
Epidermals and Glandular Elements:
Camel Hair & Dander; Cattle Hair & Dander; Cat Hair and Dander; Deer Hair & Dander; Feathers, Chicken; Feathers, Duck; Feathers, Goose;
Feathers, Parakeet; Feathers, Pigeon; Feathers, Turkey; Fox Fur;
Gerbil Hair & Epithelium; Goat Hair & Dander; Guina Pig Hair &
Dander; Hamster Hair & Epithelium; Hog Hair & Dander; Horse Hair &

~1'O 94/06821 ~ ~ ~ ~ (~ PCT/NL92/00160 Dander; Human Dander; Monkey Hair & Epithelium; Mouse Hair &
Epithelium; Dog Breeds Hair & Dander; Pyrethrum; Rabbit Hair &
Epithelium; Rat Hair & Epithelium;
Dust and Miscellaneous Extracts:
Coconut Fiber; Cotton Linters; Cottonseed; Dust, Barley; Dust, Corn;
Dust, House; Dust, Grain Mill; Dust, Mattress; Dust, Oat; Dust, Pea;
Dust, Rye; Dust, Soybean; Dust, Upholstery; Dust, Wheat; Dust, Wood-Cedar/Juniper; Dust, Wood-Fir/Hemlock; Dust, Wood-Gum; Dust, Wood-Mahogony; Dust, Wood-Maple; Dust, Wood-Oak Mix; Dust, Wood-Pine Mix;
Dust, Wood-Redwood; Dust, Wood-Spruce; Dust, Wood-Walnut; Fern Spores sp.; Flax Fiber, Flaxseed; Hemp; Jute; Kapok; Karaya Gum;
Lycopodium; Orris Root, Pyrethrum; Silk; Sisal; Tobacco; Soybean;
Castor bean;
Insect Extracts:
Ant, (Black and Red); Ants, Carpenter; Ants, Fire; Blakfly; Butter fly; Caddis Fly; Cricket; Cockroach; Deer Fly; Flea antigen;, Fruit Flies; Gnat sp.; House Fly; Mayfly sp.; Mite (D. ~artnae, D.
pteronysstmus, Leptdagt~phus spp.); Moth.
The present invention also relates to the use of the purified extracts obtainable according to the invention, for standardization, diagnosis, synthesis, and vaccination purposes.
Specific embodiments of the present invention are illustrated by the following procedures A-C.
Procedure A
Pollen granules are collected from botanically identified plants and dried in air at ambient temperature. Lipids, fatty acids, free flavonoids and other apolar free organic substances are then removed from the dry pollen by continuous extraction in a Soxhlet apparatus with organic solvents non-miscible with water, e.g. dry diethylether or n-hexane. The defatted pollen mass is again dried in air and subsequently extracted for 2 hours under mechanical agitation at a temperature between 4-20~C with aqueous solvents, i.e. dilute buffer or ammonium bicarbonate solutions, or with distilled water at a pH-value maintained between 6-8.5. The mixture WO g4J06821 pC'f/NL9?.l~lb8 is then centrifuged for 30 minutes at about 300x-5006 r.p.m., and the supernatant fluid is aolleoted. "!'he insoluble residue is dis-carded or may be re-extracted once pore by the same prxodure. The fcoaabined) aqueous extrset(s~ :ts olarified by filtration through 5 ordinary filter paper and then diel.ysed for 18 h against several changes of distilled water with a pH-vs3ue no lower than 5;~-6.0 grad ho higher then pH ~~5. For the dialysis step use 3s grade at commercial membranes vith a nominal cutoff of 5-IO kD ~~e.g. Yis~kirsgx cellophane dialysis tubing) or, alternatively, the extract nay be 10 diafiltered through suitable m~mbr~es of tt~e same cut~aff- ret~ge (e.g. Amicon ar Id3liipore Ultra- ar Di,aF~it~r-I~embrsnes). The aon dielysable retentate solution, containing the high molecular (I~) allergenic proteins is finally taken to dryness by lyophilisation, or processed directly from solution for further purification pf the i5 allergenic proteins a~' M>5'10 kD.
hn the present procedure the lyophilised materiel H~IW is redissoived in distilled water to s eoncetrtretian of 0.5-i.0 ~ w/v end the pH of the solution is sd~usted to pH a by the drvpwise additions a!' ~ N( ar more concentrated) HCI. ~'he extract is then re~
dialysed for ~~ h at s temperature between 4-~O~C against a00 volumes of distilled water (pH 6-7=5) ss the outer Iiauid. During this process the outer liquid is kept under constant agitation by placing the vessel holding both the outer liquid and the ~'ree-flaating dialysis bags on a wagnetic stirrer. After terminating the Z5 trar~s-membrane Passage of the desorbed pigmeat~, the pit~value pf the outer fluid has risen to about pH 3.5, It was fpund that the use of acidified water at pH ~ as the outer liquid directly st the start of the process does not improve the efficiency of the release of adsorbed pi&met~ts, After the separation process, the retentate fluf~d inside the dialysis bag or retained by the diafilter aeabrar~e is brought to pH 6.5-'.5 under stirring by the dropwise addition of l N
NaOH. The thus neutr~i.ised solution is fins:liy dried by lyaphili-ration to recover the depigmented ailergenie pollen proteins DPP as end product. As discussed in Example III, it was shop in the present procedure that the Iowerin; ot' the p1~ by itself does not seriously impede the arstibody recognitfon sites of the allere~enic proteins.
*trade marks ~WO 94/06821 ~ ~ ~' ~ PGT/NL92/00160 Depending on the nature and species of the pollen grains, the procedure outlined above removes 15-65 x w/w of adsorbed pigments relative the dry weight of the orginal allergenic pollen protein preparation HMW (table I). The desorbed pigment material, which in some cases also contains small peptides, may be recovered separately by concentrating the neutralised outer liquid under reduced pressure in a rotating thin-film evaporating apparatus.
Table I.
Recoveries of depigmented pollen proteins of M > 10 kD from proteins predialysed at M > 10 kD nominal cut-off after removal of adsorbed pigments and compounds of less than M = 10 kD, together with the percentages of desorbed molecules of M < 10 kD.
Pollen x yield depigmented x yield desorbed proteins M>10 kD compounds M<10 kD
Lolium perenne 67 n.d.

Dactylis glomerata 68 17 Artemisia vulgaris 63 18 Betula alba 31 64 Chenopodium album 57 36 Olea europea 31 52 Ambrosia elatior 67 n.d.

Parietaria judaica 67 15 The process is controlled by ultraviolet absorption spectroscopy. As shown in Figure 1, the extinction values at 260-280 nm of aqueous solutions of classical pollen proteins HMW, pre-dialysed at neutral pH but before acid desorption and re-dialysis, may achieve very high values, especially with the non-depigmented allergenic proteins of the pollens of the trees, weeds and shrubs.
Estimates of the protein content from the extinction coefficients at 260-280 nm in many cases therefore leads to overestimates. The removal of adsorbed pigments causes a significant drop in the extinction coefficients in the 260-280 nm range as well as at 340-360 nm, the major absorption range of the flavonols (Table II).

WO 94/06821 PCT/NL92/00~

~~~~1~~ 12 Table II

Changes in W-extinction coefficients (lx,1 cm) pH2 (HC1) E at before and after depigmentation by aciddialysis.

Pollen preparation 260 350 nm nm before after x before after Lolium perenne 7.6 9.5 +

Dactylis glomerata 10.0 5.9 -Artemisia vulgaris 26.4 22.8 -Betula alba 32.4 19.4 - 20.4 6.5 - 68 Chenopodium album 42.4 35.1 - 26.8 21.0 22 Olea europea 66.0 50.3 - 42.0 27.1 35 Ambrosia elatior 82.4 34.8 - 64.4 22.1 66 Parietaria ~udaica 108.3 103.8 - 64.8 70.4 8

4 +

The allergenic potency of the final depigmented product DPP
is compared with that of the original pollen proteins HMW by inhibition of the binding of specific IgE- or IgG-class antibodies in the blood serum of specifically allergic pollinosis patients. The IgE-binding potency of the DPP product relative to the HMW
preparation may for example be quantitatively determined by the chemical coupling of either the HMW or the DPP product to cellulose discs with cyanogen bromide or other coupling agents, followed by IgE antibody assay according to the established procedures of radioallergosorbent- or enzyme-allergosorbent tests. For quantitat-ively evaluating the reaction with specific human IgG-antibodies, either the DPP or the HMW proteins may be adsorbed physically to the surface of the wells of polystyrene microtiter plates, followed by established procedures of enzyme immunoassay.
Procedure B
In a modification of the production process, the allergenic proteins HMW are prepared from pollen granules as described in Procedure A, but the retentate solution at pH 5~5-7~5 containing the ~VfO 94/06821 ~ ~ ~ ~ PGT/NL92/00160 1~, allergenic proteins of M > 10 kD is not dried by lyophilization.
Instead, the protein solution is directly brought to pH 2 by the dropwise addition of 6 N HCL and the thus acidified solution is submitted to re-dialysis for 6-24 hours or to renewed ultrafiltration through membranes of M = 10 kD nominal cut-off. The nondialysable retentate solution is then recovered, adjusted with 1 N NaOH to pH 6.5-7.5 and dried by lyophilization.
Procedure C
The experi~vnce gained in the development of Procedures A and B of the invention demonstrates that the firm adsorption of flavonoid- or flavonoid-glycoside pigments to pollen proteins is largely due to electrostatic forces, which can be overcome for purposes of desorption by discharging negative carboxyl- and/or phenolic hydroxyl groups of either partner at a pH-value below 3Ø
However, in case such conditions are considered undesirable for considerations of possible denaturation or loss of essential struc tural protein determinants, a procedure was developed based on the elimination of adsorbed pigments in an electric field in the neutral pH-range of 6.5-7.5.
In this method, a 2-5 x solution is made of the lyophilised allergenic proteins HMW described in Procedure A, in a 0.01 M in-organic buffer salt solution pH 6.5-7.5, for example a phosphate-buffered saline solution. A suitable volume of this solution, depending on the technical equipment chosen, is then sub3ected to free electrophoresis to disrupt the ionic forces causing protein pigment adsorption. During the electrophoresis the pigments rapidly move to the cathode compartment and may thus be separated from the slow-moving protein constituents, which remain on the anodic side. A
technical prototype of this Procedure is given in Example IV.
Example I
In this experiment the dry pollen of short ragweed pollen, Ambrosia etattor (obtained commercially from Beecham Research Laboratories, England) were defatted with diethylether and extracted with distilled water as described in Procedure A. Of the dialysed and lyophilized HMW ragweed pollen protein preparation, a sample of WO 94/06821 PCT/NL92/001'~

25 mg was dissolved in 5 ml distilled water and the solution was brought to pH 2 by the dropwise addition of 6 N HC1. The ultraviolet absorption spectrum was separately observed at 1:20 dilution in 0.01 N HC1. The sample was dialysed under stirring and at ambient temperature for 24 hours against a total volume of 500 ml of distilled water. After the acid dialysis step, the UV-absorption spectrum of the inner retentate fluid was again observed at 1:2-dilution in 0.01 N HC1. The outer liquid was concentrated to the original volume of 5 ml in a RotaVapor R thin-film evaporator and the W-absorption spectrum measured in 1:20 dilution at pH 2. The successful removal of the adsorbed flavonoid pigments in this experiment is clearly demonstrated in the W-absorption spectra recorded in Figure 2 and the numerical extinction coefficients in Table II. The yield of lyophilized depigmented protein material DPP
recovered according to Procedure A is listed in Table I.
Example II
In these experiments, the same procedure as described in Example I was followed with the pollen of LoZtum perenne and Dactytts gtomezata as representative examples of potent allergenic pollens of the botanical family of the Graminese, and of Chereopodium atbum and Artemtsta tnctgarts as well-known representatives of allergenic pollens of weeds. The numerical data with respect to yields of DPP from HMW as well as the pertinent spectroscopic figures are listed in Tables I and II. The results of these experiments show that the desorption of flavonoid (-glycoside) pig ments from traditionally prepared allergenic pollen proteins HMW not only applies to pollen of the weed Ambrosia etattor as reported in Example I, but extends to other weeds as well as to the pollen of the grasses.
Example III
In these experiments, the same procedure A as described in Examples I and II was followed with the potent allergenic pollens of the trees BetuZa atba, OZea europea and of the widespread Medi terranean weed PaTtetaria ~udaica. The original allergenic proteins HMW and the corresponding depigmented proteins DPP were examined for ~WO 94/06821 , 15 , : ~ ~ ~ ~ ~ ~ PCT/NL92/00160 in vitro allergenicity by means of the inhibition of binding of specific IgE- and IgG-antibodies in the blood serum of specifically allergic patients.
For the inhibition of binding to specific IgE antibodies an established method of RAST-inhibition was chosen. In this method, a serum sample of a patient with pollinosis due to the pollen proteins to be investigated is incubated with a cellulose disc to which either the pollen proteins HMW or the depigmented counterparts DPP
have been covalently bound with the aid of cysnogen bromide or another suitable chemical coupling agent. After the capture of specific IgE-antibodies during this incubation phase, the discs are washed in a dilute buffer solution, followed by an incubation step with an enzyme-labelled anti-IgE-antibody, and the colour is finally developed with an enzyme-specific chromogenic substrate. For the evaluation of the IgE-binding potency of a given allergenic protein, sequential dilutions of the allergen are preincubated with a fixed volume of the human serum sample before the capture of residual IgE
by the allergen-coated cellulose disc. The IgE-binding potency of the allergenic preparation is read as the point of 50 x inhibition from a plot of allergen concentration versus IgE-binding, as shown for the example of BetuZa atba pollen in Figure 3. In the present Example use was made of two kinds of allergen-coated cellulose discs, viz. discs coupled chemically to the original protein preparation HMW (pigm discs in Table III) and discs coupled chemically to the protein preparations DPP prepared according to Procedure A (depigm discs in Table III) . As shown by the collected data in Table III, the allergenic IgE-binding potency of the DPP-proteins tends to decrease slightly, though not significantly, per ug of lyophilized material in the case of BetuZa atba and Parietarta ,~urlatca, whereas in the case of the Otea europea sample it must be concluded that some loss of IgE-binding epitopes indeed occurs during depigmentation.
It was checked in a separate experiment whether perhaps keeping the Olea HMW proteins at pH 2 by itself had a denaturing effect on the Otea europea pollen proteins. To this end, Olea HMW
was brought to pH 2 with HC1 according to procedure A and the solution was left standing for 4 hours at room temperature. The WO 94/06821 ~ PCT/NL92/001~
. 16 ,' solution was then readjusted to pH 7.0 without any further dialysis.
The preparation was lyophilized and checked for IgE-binding potency by BAST-inhibition, using Olea DPP discs. the results under these conditions of assay and with the particular human serum chosen were:
50 x ~T-inhibition for Olea HMW 0.75ug, Olea HMW treated at pH 2 and readjusted to pH 7 without dialysis: 0.75 ug, Olea DPP prepared according to procedure A: 3.89 ug. Hence, the decrease of IgE-binding allergenicity of Olea pollen DPP relative to Olea HMW is not due to protein denaturation at acid pH, but to removable pigments or other electrostatically adsorbed low-molecular weight organic compounds acting as true antigenic determinants. Comparative studies along these Lines underline the usefulness of depigmented pollen proteins for in-depth studies of the molecular structure of anti-body-binding allergenic epitopes.
For the inhibition of binding to specific IgG antibodies an established method of enzyme immunoassay was chosen. In this method, a serum sample of a patient with pollinosis due to the pollen proteins to be investigated is incubated with sequential dilutions of the HMW or DPP allergen. The allergen-serum mixtures are then pipetted into the wells of polystyrene microtiter plates pre-coated with either the HMW or DPP proteins by physical adsorption. After 30 minutes at room temperature, the wells are then washed with dilute buffer solution and the specific IgG antibody captured on the allergen-coated plate is determined by treatment with an enzyme-labelled anti IgG-antiserum, followed by colour development with an enzyme-specific chromogenic substrate. The IgG-binding potency of the allergen preparation is evaluated as the point of 50 inhibition interpolated on the plot of allergen concentration versus IgG-binding. In the experiments of this Example III, the microtiter wells were in all cases coated with the DPP-preparations produced according to procedure A. The data in Table III show that the potency of the DPP in IgG-binding increases slightly relative to the HMW products.

~VO 94/06821 2 ~ 4 JC ~ ~ ~ PCT/NL92/00160 Table III
Potency of DPP pollen proteins relative to traditional non-depigmented pollen proteins HMW for binding of specific IgE-(by RAST) and IgG-antibodies (by EIA). Figures given represent ug of preparation required for 50 x of antibody binding in the test system chosen.
Allergen RAST
Pigm Depigm EIA for IgG
discs discs (DPP-coating) HMW DPP HMW DPP HMW DPP

Betula 0.108 0.071 0.24 0.12 46.0 17.2 Olea 0.163 1.31 0.75 4.02 0.12 0.05 Parietaria 0.18 0.31 2.3 1.77 0.11 0.001 F~xsmDle IV
In these experiments the original pollen proteins HMW of ragweed (Ambrosia etatior) isolated and dialysed at neutral pH were depigmented by free electrophoresis according to procedure C. A
solution of 20 mg HMW/ml was made and 5 ml of this solution was brought into a cellophane dialysis bag (Visking dialysis tubing, nominal cut-off 10000 D) . The bag was sealed both ends and brought into the rotating plastic holder of the electrophoresis equipment RotaPhor R (BioRad, USA). The void volume of the holder tube was filled with a phosphate buffered-saline solution (0.01 M, pH 7.4) and the equipment was put into a cold room at 4~C. Electrophoresis was then started by applying a DC electric potential at a constant power of 12 Watts under continuous rotation of the tube holding the dialysis bag and the outer liquid. The current drupped from 120 mA
at the start of the experiment to 68 mA after 3 hours. During the electrophoretic run, the pigments are released from the pollen proteins and migrate out of the dialysis bag to the cathodic com-partment of the outer liquid (negative pole). After the run, the depigmented proteins were recovered from the solution inside the dialysis bag and dried by lyophilization. The recovery of DPP

WO 94/06821 ~ ~ C~' ~ ~ PGT/NL92/00~

product from 100 mg of Ambrosia eZatior HMW was 69 mg, representing a 69 x yield of depigmented material by electrophoresis at neutral pH according to procedure C, as compared to 67 x according to procedure A (Table I).

Claims (9)

1. A process for obtaining aqueous extracts containing allergenically active proteins which are substantially free of adherence to undesirable compounds comprising the steps of a) preparing an aqueous extract containing allergenically active proteins to which undesirable non-allergenic compounds are adhered by electrostatic, hydrophobic or other physical forces;
b) disrupting the electrostatic, hydrophobic or other physical forces under such conditions as to disadhere said non-allergenic compounds from said allergenically active proteins; and c) collecting the resulting aqueous extract, containing allergenically active proteins which are substantially free from adherence to non allergenic compounds.

2. The process according to claim 1, wherein the non-allergenic compounds have a molecular weight of less than 10,000 daltons.

3. The process according to claim 1, wherein the non-allergenic compounds comprise at least one compound selected from the group consisting of flavonoids and glycosides of flavonoids.

4. The process according to claim 1, wherein the non-allergenic compounds have a molecular weight of less than 5,000 daltons.

5. The process according to claim 1, wherein the electrostatic, hydrophobic or other physical forces are disrupted by at least one chemical selected from the group consisting of said, alkaline materials including anion- and cation-exchanging materials, salts and electric currents.

6. The process according to claim 5, wherein the and and alkaline chemicals are used in an amount sufficient to tease an iso-electric paint higher than an iso-electric point of said proteins.

7. The process according to claim 5, wherein the electrostatic force is disrupted by an acid having a pH-value of less than 3.

8. The process according to claim 5, wherein the electrostatic force is disrupted by electric currents in the form of electrophoresis.

9. The process according to claim 5, wherein the electrotatic force is disrupted by an acid having a pH-value of 1.5-2.5.