Method for producing hypoallergenic major birch pollen allergen rbet v 1

Abstract

The invention relates to a process for the preparation of hypoallergenic major birch pollen allergens by means of one or more chromatograpluc purification steps using essentially unbuffered aqueous bases as eluent and subsequent neutralisation. The hypoallergenic major birch pollen allergens are distinguished by a lack of, but at least by reduced immunoglobulin E binding with simultaneous retention of therapeutically relevant T-cell stimulation. They can therefore be employed as low-side-effect therapeutic agents for specific immunotherapy.

1 . Process for the preparation of hypoallergenic major birch pollen allergen rBet v 1 by means of one or more chromatographic purification steps using essentially unbuffered aqueous bases as eluent and subsequent neutralisation, characterised in that the starting material employed is a soluble rBet v 1 crude protein prepared by recombinant methods.  
     
     
         2 . Process according to  claim 1 , characterised in that the soluble rBet v 1 crude protein prepared by recombinant methods was expressed in  E. coli.    
     
     
         3 . Process according to  claim 1 , characterised in that the chromatographic purification steps include anion exchange chromatography, hydrophobic interaction chromatography and gel filtration.  
     
     
         4 . Process according to  claim 1 , characterised in that the chromatographic purification steps are carried out in the following sequence: first gel filtration, anion exchange chromatography, hydrophobic interaction chromatography, second gel filtration.  
     
     
         5 . Process according to  claim 1 , characterised in that the base used as eluent is employed in a concentration range from 5 mM to 100 mM, preferably from 5 mM to 40 mM.  
     
     
         6 . Process according to  claim 1 , characterised in that the base used as eluent is NaOH.  
     
     
         7 . Process according to  claim 1 , characterised in that an inorganic neutral salt and optionally NaHCO 3  are added to the eluent.  
     
     
         8 . Process according to  claim 1 , characterised in that the inorganic neutral salt added to the eluent is NaCl.  
     
     
         9 . Process according to  claim 6 , characterised in that the concentrations of the substances NaOH, NaCl and NaHCO 3  present in the eluent are selected in such a way that the typical concentrations for physiological saline solution can be set in the neutralisation step following the purification.  
     
     
         10 . Process according to  claim 1 , characterised in that the rBet v 1 crude protein is pre-purified in a buffered solution by chromatography and/or by salt precipitation.  
     
     
         11 . Process for reducing the IgE activity of major birch pollen allergen rBet v 1, characterised in that a soluble major birch pollen allergen rBet v 1 prepared by recombinant methods is subjected to one of the processes described in  claim 1 .  
     
     
         12 . Hypoallergenic major birch pollen allergen rBet v 1 obtainable by one of the processes described in  claim 1 .  
     
     
         13 . Hypoallergenic major birch pollen allergen rBet v 1 according to  claim 12  as medicament.  
     
     
         14 . Use of hypoallergenic major birch pollen allergen rBet v according to  claim 12  and/or pharmaceutically usable derivatives thereof, including mixtures thereof in all ratios, for the preparation of a medicament for the specific immunotherapy of allergies in the initiation of which major birch pollen allergen Bet v 1 is involved.  
     
     
         15 . Pharmaceutical composition comprising a hypoallergenic major birch pollen allergen rBet v 1 according to  claim 12  and/or pharmaceutically usable derivatives thereof, including mixtures thereof in all ratios, and optionally excipients and/or adjuvants.