Inhibitors, or neutralizing antibodies against coagulation factor VIII (FVIII), hinder FVIII replacement therapy in congenital hemophilia A and can also arise as autoantibodies in acquired hemophilia A. The development of inhibitors is a major cause of morbidity and mortality in congenital hemophilia A. However, the circulating anti-FVIII antibody compartment in humans remains poorly understood, hindering the development of novel treatments for patients with inhibitors. For the first time, we have developed and fully characterized a workflow for the unbiased capture and purification of anti-FVIII antibodies, including inhibitory antibodies, from human plasma for downstream immunoproteomic studies. Using biotinylated FVIII coupled to magnetic streptavidin beads, our protocol was able to capture &gt;99% of anti-FVIII IgG from the plasma of two high-titer inhibitor patients, enriching the anti-FVIII IgG up to 400-fold. We were also able to capture all 12 monoclonal antibodies tested targeting all five immunogenic domains of FVIII, indicating a negligible effect of biotinylation on antibody binding. Furthermore, cleavage of the biotin linker demonstrated complete elution of FVIII-specific IgG from the beads. The anti-FVIII purity of the elutions was estimated to be &gt;80% after one purification and &gt;95% after a second purification. Purification had negligible effects on the FVIII-binding activity of anti-FVIII IgG in ELISA and the inhibitory activity of inhibitors in Bethesda assay. Additionally, we were able to perform peptide mapping of two monoclonal anti-FVIII antibodies by LC-MS/MS with 10 fmol and 1 pmol in the input, respectively, indicating our purification protocol is unlikely to limit the sensitivity of downstream assays. In summary, our magnetic bead-based workflow is faster and at least equally as efficient as conventional affinity purification methods, unbiased, highly specific, and easily scalable.

Highly efficient and unbiased isolation of anti-factor VIII antibodies from hemophilia A inhibitor patient plasma

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.