Allergen regulation in the future : what will be the place for recombinant allergens ?

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
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

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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
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

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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

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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

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Quality control of allergens extracts use of
recombinant allergens as markers
Recombinant allergens
pollen SDS-PAGE - Allergens from Phleum pratense
(Gramineae) extracts and recombinant markers
Phl p2
Bet v2
Phl p1
Phl p3
MW
STD NIBSC
IHR
STD NIBSC
Mfr 2
STD NIBSC
Mfr 3
MW
MW
Source AFSSAPS/DLC, Biotechnology, Protein
Biochemistry and Macromolecules Unit (Agnès
Bertocchi, Chantal Truzman, Gilles Chaudemanche)
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Quality control of allergens identification
through mass spectrometry
Birch pollen recombinant bet v1
Birch pollen commercial extract
Source AFSSAPS/DLC, Biotechnology, Protein
Biochemistry and Macromolecules Unit (Gilles
Chaudemanche Agnès Bertocchi,)
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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

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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

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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

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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