Representative superimposed sensorgrams showing single-cycle kinetics experiments in which WT-FVIII-C2 and FVIII-C2 muteins were injected at 5 increasing concentrations over biosensor flow channels with captured murine anti-FVIII mAbs, as indicated. Residues were flagged as potential contributors to the epitope if the k_d for the FVIII-C2 mutein was >2.0× the k_d for the WT protein. (A) Alanine substitutions at residues E2228, L2252, S2254, H2315, and Q2316 met this criterion in this SPR run with mAb 1B5 (which is a type B inhibitor). Separate SPR runs identified residues F2196, T2197, N2198, F2200, T2202, R2220, Q2222, N2225, and K2239 (subsequently identified as an outlier) as also possibly contributing to the epitope recognized by mAb IB5. (B) WT-FVIII-C2 and FVIII-C2 muteins Y2195A, Q2213A, N2225A, Q2270A, and T2272A were injected over the flow channel containing mAb I54 (which is a type A inhibitor). The substitution Q2213A abrogated binding to this mAb, indicating that Q2213 forms part of the epitope recognized by I54. (C) WT-FVIII-C2 and FVIII-C2 muteins Y2195A, Q2213A, N2225A, Q2270A, and T2272A were injected over the flow channel in a second SPR run to analyze interactions of FVIII-C2 muteins with mAb 1B5. Altered binding kinetics indicated that residue N2225 forms part of the epitope recognized by 1B5. (D) WT-FVIII-C2 and FVIII-C2 muteins Y2195A, Q2213A, N2225A, Q2270A, and T2272A were injected over the flow channel containing mAb 2-117 (which is a type C inhibitor). Altered binding kinetics indicated that residues Q2270 and T2272 form part of the epitope recognized by 2-117.