A rapid method to reduce drug interferences for antibody measurements in pegunigalsidase alfa-treated patients with Fabry disease

Aim: Neutralizing anti-drug antibodies (ADA) limit therapy efficacies significantly. Pegunigalsidase alfa is a newly approved drug for the treatment of Fabry disease. The increased plasma half-life due to PEGylation interferes with ADA measurements including ELISAs and serum-mediated inhibition assays. We developed a rapid protocol eliminating pegunigalsidase alfa from blood samples, without interfering downstream applications. Methods: A rapid protocol based on alkaline-pretreatment followed by acid-based neutralization of agalsidase-spiked control sera was established. Results were confirmed using serum samples from patients with and without neutralizing ADAs drawn during infusions. Repeated ADA measurements including serum-mediated inhibition assays and ELISA-based immunoglobulin isotyping (IgG, IgA, IgM) were performed with sera from 17 patients receiving pegunigalsidase alfa. Results: formation of ADAs against pegunigalsidase alfa was identified. Immunoglobulin isotyping showed mainly IgM antibodies towards pegunigalsidase alfa, recognizing PEG moieties and amino acids in this patient. Although his ADAs had a low inhibitory capacity, Western blot analyses demonstrated that the reduced pharmacokinetics might be linked to leucocyte-mediated enzyme elimination. A second patient with pre-existing ADAs before pegunigalsidase alfa-initiation showed a massive induction of anti-PEG antibodies with inhibitory function. Conclusion: We present a rapid alkaline-treatment based method to overcome drug interferences to measure at least free antibodies in patients treated with pegunigalsidase alfa.