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U.S. Department of Health and Human Services

Vaccines, Blood & Biologics

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2/15/2007 Telecon Information Request - RECOTHROM

TO: ZymoGenetics, Inc.
RE: STN 125248.0 Original Application Thrombin (Recombinant)

Request for additional information:

Regarding the validation report for the immunogenicity assays:

  1. The cut-off absorbance values for positive screening results were chosen such that 90-95% of samples from untreated normal volunteers would be negative. Please describe the number and selection criteria for the untreated normal volunteers and submit the data used to select the cut-off values. Were the 5-10% of positive samples further assessed to determine their titer? It would appear that any true positive samples should have been eliminated from the data set used for setting the cut-off value. Please comment.

     

     

  2. In the ELISA procedures, the "recommended" test sample dilution for Tier 1 was 1:50. In the Tier 2 analysis of positive Tier 1 samples, sample dilutions were 1:50, 1:150, 1:450, and 1:1350. It would thus appear that samples with antibody titers less than 50 would not be reported as positive. Please repeat the assays using a lower initial dilution, as low as feasible. While the data do show that fewer subjects in the test group developed high-titer antibodies than in the control group, the consequences of developing low-titer antibodies to r-Thrombin are unknown. There could be very different implications for repeat exposure depending on whether a patient developed no antibodies or developed low-titer antibodies after first exposure.

     

     

  3. The validation report does not assess relative sensitivities of the ELISAs for antibodies to bovine or recombinant thrombin. Please explain why the non-specific background samples were different, i.e., --------------------------------------- for the recombinant thrombin ELISA, ------------------------------------------- for the bovine thrombin ELISA. In repeating the assays as requested above, it is recommended that the two ELISAs be as similar as possible; ideally, only the antigens and positive controls would be different. Positive antibody controls were raised in --------. -------- antibodies to bovine thrombin provided higher absorbance signals at lower concentrations than -------- antibodies to recombinant thrombin. Please provide data for each ELISA that demonstrate the plateau of absorbance. It could well be that the titers are higher for ------------- anti-bovine than for ------- anti-recombinant; but if the absorbances at plateau are different, it could also be that antigen coatings for the two ELISAs are different, leading to differing sensitivities. It is recommended that the two ELISAs should provide similar absorbances at plateau, as well as similar negative control absorbances.

     

END

 

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