Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Plant proteins – e.g. – derived from legumes – algae or...
Patent
1995-08-14
1998-06-23
Tsang, Cecilia J.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Plant proteins, e.g., derived from legumes, algae or...
530370, 530372, 530412, 530416, 530417, 530427, A61K 3578, C07K 1400
Patent
active
057706980
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
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 injections for long-term "desensitization", "hyposensitization", or "immunotherapy". Based on the observation that the causative major 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.
2. Description of Related Art
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 molecular weight of about 10 kD ( Moore M B and Moore E E. J Am Chem Soc 1931; 53: 2744; Unger L, Cromwell H W, Moore M B. J Allergy 1932; 3: 253).
These investigations indicated that the low-molecular constituents of M<5 kD from pollen extracts do indeed exhibit some residual allergenic activity, although their potency on a weight basis is a factor of at least 1000 less than that of the non-dialysable 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 pursuing 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 nevertheless remained ambiguous. It is usually tacitly assumed that such compounds, which may considerably influence cellular functions 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 FIG. 1).
SUMMARY OF THE INVENTION
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.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts 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.
FIG. 2 depicts the results of Example I, where the process claimed in the invention was applied to Ambrosia elatior. FIG. 2 projects the successful removal
REFERENCES:
Guerin et al, J. Immunol. Methods, vol. 55, pp.265-271, 1982.
Marcy, Chemical Abstracts, vol. 83, p. 370, Ref. #81847f, 1975 (Fr. Patent No. 2,228,070, Nov. 29, 1974).
C.B.F. Leti S.A.
Mohamed Abdel A.
Tsang Cecilia J.
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