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Abstract

Heberprot-P® is a therapeutic product approved in Cuba for the treatment of diabetic foot ulcer. In spite of its ample clinical use, its immunogenicity in patients has not been evaluated. Regulatory agencies have developed guidelines for the validation of immunoassays designed at the detection of anti-drug antibodies to biologicals. The aim on this work was to validate a method for the detection of binding antibodies in human serum to recombinant human epidermal growth factor (rhEGF), the active ingredient of Heberprot-P®. A two-tiered enzyme-linked immunosorbent assay (ELISA) format was used. First tier was a screening assay, based on immobilized rhEGF and anti-human polyvalent alkaline-phosphatase conjugated as development reagent. Positive samples were then tested with a confirmatory assay which used a competitive soluble rhEGF in solution. As FDA and EMA guidelines recommend, minimum required dilution, quality controls, screening and confirmatory cut points, sensitivity, recovery, precision, specificity, selectivity, robustness and stability of samples were determined. The assay was precise and its sensitivity was calculated to be 214.2 ng/mL. The majority of evaluated parameters fulfilled the figures recommended by current guidelines. The immunoassays described in this work could be reliable tools for the evaluation of immunogenicity of Heberprot-P® in well-designed clinical studies.