1. Allergen regulation in the future : what will be the place for recombinant allergens ? Jacqueline DAYAN-KENIGSBERG European Academy of Allergology and Clinical Immunology, Allergy summer school on recombinant allergens, Bischenberg-Bisschoffsheim, Sept 21-24, 2007.

2. 2 28/08/2015 – Recombinant Allergens - Allergy School 2007 Allergens European regulatory framework Directive 89/342 CEE immunological medicines (vaccines, sera or allergens) European codification 2001 : Article 1 point 4 : « allergen product » shall mean any medical product which is intended to identify or induce a specific acquired alteration in the immunological response to an allergenic agent Note for guidance on allergen products CPMP/BWP/243/96 Concept paper on the revision of the note for guidance on allergen products CHMP/BWP/229472/2005

3. 3 28/08/2015 – Recombinant Allergens - Allergy School 2007 Quality requirements (active substance) General information Standardisation important Recombinant allergens consist of predefined allergenic polypeptides e.g. major allergen or mixture of defined polypeptides Quantity and structure of these polypeptides can be determined These products should be standardized as other biological products consisting of purified proteins

4. 4 28/08/2015 – Recombinant Allergens - Allergy School 2007 Quality requirements (active substance) Guidelines for products derived from DNA technology : Q5B (CPMP/ICH/139/95) : analysis of the expression construct in cell lines used for production of rDNA-derived protein products Q5C (CPMP/ICH/138/95): stability testing of biotechnological / biological products Q5D (CPMP/ICH/294/95): derivation and characterisation of cell substrate used for production of biotechnological / biological products Q5E (CPMP/ICH/5721/03): comparability of biotechnological / biological products Q6B (CPMP/ICH/365/96): test procedures and acceptance criteria for biotechnological / biological products

5. 5 28/08/2015 – Recombinant Allergens - Allergy School 2007 Quality requirements (active substance) Characterisation and control of recombinant allergens Characterisation and quantification by techniques appropriate for recombinant proteins Content expressed in weight per volume Correlation between quantity of individual recombinant allergen and biological activity validated ELISA methods with specific animal antibodies may be used as potency assays if correlation with IgE binding has been demonstrated For mixtures of different allergens, contents of individual allergens to be determined prior to mixing

6. 6 28/08/2015 – Recombinant Allergens - Allergy School 2007 Quality requirements (finished product) Control All tests for batch release should be performed on the finished product whenever possible If any of the control tests (e.g. potency tests) cannot be performed on the finished product, quality specifications should be defined for the intermediate at the latest stage prior to the modification test Content of the purified protein (major allergen) and the potency should be indicated Special attention should be given on impurities by media or host cell components. These impurities should be identified and quantified and their eventual undesirable allergenic potential estimated Stability testing should be performed as real time stability studies

7. 7 28/08/2015 – Recombinant Allergens - Allergy School 2007 MW STD NIBSC IHR STD NIBSC Mfr 2 STD NIBSC Mfr 3MW Recombinant allergens Phl p1 Phl p2 Phl p3 Bet v2 MWManufacturer 1 pollen SDS-PAGE - Allergens from Phleum pratense (Gramineae) extracts and recombinant markers Quality control of allergens extracts : use of recombinant allergens as markers Source : AFSSAPS/DLC, Biotechnology, Protein Biochemistry and Macromolecules Unit (Agnès Bertocchi, Chantal Truzman, Gilles Chaudemanche)

8. 8 28/08/2015 – Recombinant Allergens - Allergy School 2007 Birch pollen recombinant bet v1 Quality control of allergens : identification through mass spectrometry Birch pollen commercial extract Source : AFSSAPS/DLC, Biotechnology, Protein Biochemistry and Macromolecules Unit (Gilles Chaudemanche Agnès Bertocchi,)

9. 9 28/08/2015 – Recombinant Allergens - Allergy School 2007 Recombinant allergens : interest (1) Reproducible production in high quantities Improvement of extracts used for diagnosis: by selection of the most relevant allergenic sources by quantification of their major allergen content Molecular basis of cross-reactivity between inhaled and/or food allergens Quantitative evaluation of IgE responses

10. 10 28/08/2015 – Recombinant Allergens - Allergy School 2007 Recombinant allergens : interest (2) Extracts with constant allergenic activity Standardisation of allergen extracts made easier Extracts containing only relevant allergens Elimination of contaminating proteins (other allergens, non-allergenic proteins) Reduction of contamination by viral or infectious agents Possibility to produce derived recombinant allergens with reduced allergenic activity to reduce adverse reactions

11. 11 28/08/2015 – Recombinant Allergens - Allergy School 2007 Recombinant allergens : what do we need ? Sera or cells from patients sensitised to natural allergens to compare immunological activity of recombinant and natural allergens Clinical study design must target appropriate patient population for greatest therapeutic effect Correlation between major allergen concentration and biological activity Evidence that : purity of the product is consistent correlation between biological activity and mass is defined and consistent. product and biological activity are stable under conditions of storage and shipping

12. 12 28/08/2015 – Recombinant Allergens - Allergy School 2007 Recombinant allergens : conclusion New tools for diagnosis and conventional desensitisation New european regulations Use of recombinant allergens in vivo : studies in progress to make it possible in the future Challenging perspectives : recombinant hypoallergenic allergen derivatives for specific immunotherapy tailored to sensitisation profile of allergic patients prophylactic vaccination