Recombinant human factor VIII-specific affinity ligands selected from phage-displayed combinatorial libraries of protein A

Factor VIII-specific affibodies were selected from phage displayed librarie s constructed by combinatorial mutagenesis of an alpha helical bacterial re ceptor domain derived from staphylococcal protein A. Bead-immobilized recom binant human factor VIII (rVIII) (80 and 90 kDa chains) protein was used du ring competitive biopannings in the presence of free 80-kDa chain protein, resulting in the selection of several binders that showed dissociation cons tants (K-d) in the range 100-200 nm as determined by biosensor analyses. On e variant (Z(rVIII:3), 90-kDa chain specific) was further characterized in small-scale affinity chromatography experiments, and showed efficient and s elective recovery of biologically active rVIII from Chinese hamster ovary c ell supernatant-derived feed stocks. The purity of the enriched rVIII was c omparable with rVIII material purified by immunoaffinity chromatography usi ng a 90-kDa chain-specific monoclonal antibody. Interestingly, epitope mapp ing showed that the monoclonal antibody and the affibody ligand competed fo r the same or at least overlapping epitopes on rVIII. In addition, the Z(rV III:3) variant was produced by peptide synthesis with a C-terminal cysteine to enable directed coupling to solid supports. This 59-residue protein was analyzed by circular dichroism and showed a secondary structure content si milar to that of the parental Z domain used as scaffold. In biosensor studi es. the synthetic affibody was immobilized recruiting the C-terminal cystei ne residue, and demonstrated to bind both recombinantly produced and plasma -derived factor VIII, From a secondary library, constructed by re-randomiza tion of relevant positions identified after alignment of the first-generati on variants, a panel of affinity-improved second-generation affibodies were selected of which one clone showed a dissociation constant (K-d) for rVIII of 5 nM. Several of these variants also showed higher apparent binding eff iciencies towards rVIII when analyzed as immobilized ligands in biosensor e xperiments. Taken together, the results suggest that affibody ligands produ ced by bacterial or synthetic routes could be of interest as an alternative to monoclonal antibodies in purification processes or as diagnostic or mon itoring tools.