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U.S. Food and Drug Administration
Notice: Archived Document The content in this document is provided on the FDA’s website for reference purposes only. It was current when produced, but is no longer maintained and may be outdated.
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Update on the FDA/CBER Allergen Standardization Program
Jay E. Slater, MD Division of Bacterial, Parasitic, and Allergenic Products FDA/CBER/OVRR
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Acknowledgements FDA/CBER National University of Singapore
T. Khurana, PhD S. Huynh M. Collison N. deVore, PhD J. Finlay, PhD National University of Singapore F. T. Chew, PhD University of Virginia J. Woodfolk, MD, PhD S.M. Satinover, PhD Yonsei University College of Medicine K.Y. Jeong, PhD
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Today’s presentation Regulatory basis of allergen standardization in the US Prior standardized allergens Recent research activities Future directions
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Summary of CBER’s approach
Potency measures not universally required: there are standardized and non-standardized allergen extracts For standardized allergens National reference standards National unitage ID50EAL is the preferred method for establishing potency Surrogates: Competition ELISA Specific antigen assay Mass units
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There are hundreds of non-standardized allergen extracts
Unitage is uninformative PNU/mL w/v (usually 1:10 or 1:20)
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Allergen standardization (21CFR 600.3(s), 610.10 and 680.3(e))
Potency: “the specific ability or capacity of the product, as indicated by appropriate…tests or…clinical data…to effect a given result” Potency tests: “in vivo or in vitro tests, or both…specifically designed for each product…” “The potency of each lot of each Allergenic Product shall be determined…[T]he potency test methods shall measure the allergenic activity…Until manufacturers are notified…of the existence of a potency test…[they] may continue to use unstandardized potency designations.”
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Allergen standardization (21 CFR 680.3(e))
Establish a US standard, and Establish a testing procedure Manufacturers may use the established procedure, or may develop equivalent procedures
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CBER’s allergen standardization activities
Baer Kenimer DeVries Slater Rabin retires
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Standardized products (n = 19)
D. farinae D. pteronyssinus Cat hair Cat pelt Short ragweed pollen Hymenoptera Honey bee Wasp Yellow jacket Yellow hornet White-faced hornet Mixed vespid Grass pollens Bermuda grass Red top June (Kentucky blue) Perennial rye Orchard Timothy Meadow fescue Sweet vernal
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Unitage for standardized allergens
Hymenoptera venoms - µg protein Mites - AU Grass pollens - BAU Short ragweed pollen - Amb a 1 U Cat hair and cat pelt – BAU and Fel d 1 U
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Allergen extract characterization is a rapidly evolving field
To select any unit for extract labeling, we need to address the issue of scientific advances after unitage is designated
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Scientific advances I Cat Standardized extracts approved 1988-90
Overall potency: AU/mL Determined as Fel d 1 U Subsequent advances ID50EAL testing performed Importance of cat albumin recognized in Action: designate cat hair and cat pelt; switch to BAU/mL Turkeltaub and Matthews, J Allergy Clin Immunol 1992; 89:151
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Scientific advances II
Mites Standardized extracts approved 1988+ Overall potency: AU/mL Subsequent advances ID50EAL testing done Importance of Group 1/2 allergen recognized Overall potency standards continued
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Scientific advances III
Timothy grass Standardized extracts approved 1997+ Overall potency: BAU/mL, based on ID50EAL testing Importance of specific grass pollen allergens recognized in 1980’s and 1990’s, but correlation to overall biological activity is complex
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Scientific advances IV
German cockroach Standardization candidate in US since 2001 Multiple allergens identified and well-characterized Correlation to overall biological activity still not established In vitro data suggested correlation between Bla g 1 and Bla g 2 and overall potency (competition ELISA) ID50EAL potency did not correlate with Bla g 1, Bla g 2 or Bla g 5 levels Patterson and Slater, Clin. Exp. Allergy 2002; 32: Slater, et al., Clin. Exp. Allergy 2007; 37:
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Existing solution: a flexible potency determination
Total protein (hymenoptera) Overall allergen (grasses, mites) Pooled human antibody Specific allergen (cat, ragweed) Sheep antibody
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The dilemma of these potency measures:
In order to measure specific allergens, we need to know which allergens are relevant If we measure overall allergenicity, we are unable to detect the absence of specific (and potentially important) allergens
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Specific loss of a single allergen
Soldatova et al., J Allergy Clin Immunol 2000;105:
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Two possible solutions:
Divide the signal by Separating the allergens, or Separating the antibodies
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Antibody multiplex assay
Advantages quantitative reflects spectrum of allergen recognition does not require identification of relevant allergens Disadvantage New technology; initial development will be labor intensive and expensive
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scFv attached via amide bond
Carboxylated Beads o c N O S O- C EDC + Sulfo NHS Sulfo-NHS ester scFv attached via amide bond scFv Coupling of beads with scFv
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Rabbit polyclonal sera
Assay design Streptavidin – RPE anti-rabbit Biotin Rabbit polyclonal sera scFv target in extract scFv bound to Carboxy labeled bead Luminex 200, Luminex Corp. 635 nm 532 nm Bead dye RPE
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Multiplex approach Multiple concurrent assays Output
Each individually optimized Output Pattern recognition - qualitative Quantitative Cluster analyses Parallelism
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avian scFvs to cat and ragweed allergens expressed
Multiplex microbead assay can accurately measure potency of cat and short ragweed pollen extracts avian scFvs to cat and ragweed allergens expressed multiplex microbead quantification of Fel d 1 and Amb a 1 content correlates closely with established methods Finlay et al. Clin Exp Allergy 2005; 35: DeVore et al. Ann Asthma All Immunol 2010; 105:
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Immunoblot analysis of avian scFv antibodies against German cockroach (CR) extract (E6Cg)
31 38 76 102 150 24 MW KDa
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Minimal interference among avian anti-CR antibodies 4E4, 6A3 and 6G2
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Avian anti-CR antibodies recognize mostly heat-labile epitopes in CR
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Specific targets for the avian anti-CR antibodies
Antibody Putative target Accession # Mass (kD) 2A1 Per a 3 homologue GU 78 6A2 tropomyosin (Bla g 7) AF260897 33 6A3 vitellogenin CAA06379 213 11 unidentified None to Bla g 1, Bla g 2, Bla g 4, or Bla g 5 (Woodfolk and Satinover, Charlottesville, VA)
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6A5 5D1 5D8 6A1 6A2 6A6 6B11 6B12 6E1 2A1 4B7 4E4 6A3 6G2 tropomyosin Per a 3 homolog vitellogenin 31 38 76 102 150 24 MW KDa Bla g 2 (36) Bla g 5 (23) Bla g 4,8 (21) Bla g 7 (33) Bla g 1 (46) Bla g 6 (17)
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Summary Unitage in US labeling of standardized allergen extracts is based on the best science available at licensure. Advances after licensure pose challenges for proper unitage designations. Multiplex allergen potency determinations may facilitate a more flexible approach to potency labeling in the future. This is being pursued for German cockroach allergen standardization.
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