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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

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

Today’s presentation Regulatory basis of allergen standardization in the US Prior standardized allergens Recent research activities Future directions

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

There are hundreds of non-standardized allergen extracts Unitage is uninformative PNU/mL w/v (usually 1:10 or 1:20)

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.”

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

CBER’s allergen standardization activities 88 89 90 91 92 93 94 95 96 97 98 99 00 01 02 03 04 05 06 07 08 09 10 11 Baer Kenimer DeVries Slater Rabin retires

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

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

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

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 1991-2 Action: designate cat hair and cat pelt; switch to BAU/mL Turkeltaub and Matthews, J Allergy Clin Immunol 1992; 89:151

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

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

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; 32:721-727 Slater, et al., Clin. Exp. Allergy 2007; 37: 1033-1039

Existing solution: a flexible potency determination Total protein (hymenoptera) Overall allergen (grasses, mites) Pooled human antibody Specific allergen (cat, ragweed) Sheep antibody

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

Specific loss of a single allergen Soldatova et al., J Allergy Clin Immunol 2000;105:482-488.

Two possible solutions: Divide the signal by Separating the allergens, or Separating the antibodies

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

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

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

Multiplex approach Multiple concurrent assays Output Each individually optimized Output Pattern recognition - qualitative Quantitative Cluster analyses Parallelism

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; 35:1040-8. DeVore et al. Ann Asthma All Immunol 2010; 105:351-358.

Immunoblot analysis of avian scFv antibodies against German cockroach (CR) extract (E6Cg) 31 38 76 102 150 24 MW KDa

Minimal interference among avian anti-CR antibodies 4E4, 6A3 and 6G2

Avian anti-CR antibodies recognize mostly heat-labile epitopes in CR

Specific targets for the avian anti-CR antibodies Antibody Putative target Accession # Mass (kD) 2A1 Per a 3 homologue GU086323.1 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)

6A5 5D1 5D8 6A1 6A2 6A6 6B11 6B12 6E1 2A1 4B7 4E4 6A3 6G2 tropomyosin Per a 3 homolog vitellogenin 33 78 213 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)

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.