METHODE POUR LA PURIFICATION D'EXTRAITS AQUEUX RENFERMANT DES PROTEINES ALLERGENES; EXTRAITS POUVANT ETRE OBTENUS GRACE A CETTE METHODE ET LEUR UTILISATION

A PROCESS FOR THE PURIFICATION OF AQUEOUS EXTRACTS CONTAINING ALLERGENICALLY ACTIVE PROTEINS, EXTRACTS OBTAINABLE ACCORDING TO THIS PROCESS AS WELL AS THEIR USE

Abrégé anglais

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.

Revendications

1
CLAIMS
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.

2
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.

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.

CA 02145169 2002-06-12
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~
6
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~
8
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
9
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

CA 02145169 2002-06-12
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
11
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 +
26
Dactylis glomerata 10.0 5.9 -
41
Artemisia vulgaris 26.4 22.8 -
14
Betula alba 32.4 19.4 - 20.4 6.5 - 68
40
Chenopodium album 42.4 35.1 - 26.8 21.0 22
17 -
Olea europea 66.0 50.3 - 42.0 27.1 35
23 -
Ambrosia elatior 82.4 34.8 - 64.4 22.1 66
58 -
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'~
14
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
17
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
20
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~
18
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).