File S1 - A Single Chain Variant of Factor VIII Fc Fusion Protein Retains Normal In Vivo Efficacy but Exhibits Altered In Vitro Activity

Figures S1–S5 and Table S1. Figure S1. Mass spectra of 6 kDa LC N-terminus peptide fragment of FVIII variants after thrombin digestion and LC-MS (TOF) analysis. (a) rFVIIIFc (b) BDD rFVIII (c) SC rFVIIIFc (d) SC rFVIIIFc R1645A/R1648A. Major digestion products are indicated. Figure S2. Deconvoluted spectra of A2 fragment of FVIII variants after thrombin digestion and LC-MS (TOF) analysis. (a) rFVIIIFc (b) BDD rFVIII (c) SC rFVIIIFc (d) SC rFVIIIFc R1645A/R1648A. Major digestion products are indicated. Figure S3. Representative SPR sensorgrams of interactions between FVIII variants and VWF. One representative graph is displayed for (A) rFVIIIFc and (B) SC rFVIIIFc, respectively. Black indicates the binding curve and red indicates the best fit to a 1∶1 interaction model. Figure S4. Thrombin-mediated release of activated FVIII variants from VWF at 25°C. (A) Single reference subtracted sensorgrams. (B) Double reference subtracted sensorgrams for the phase corresponding to thrombin application. (C) Thrombin-mediated release rate as a function of time. (D) Peak thrombin-mediated release rate as a function of thrombin concentration. Figure S5. Thrombin-mediated release of activated FVIII variants from VWF at 37°C. (A) Single reference subtracted sensorgrams. (B) Double reference subtracted sensorgrams for the phase corresponding to thrombin application. (C) Thrombin-mediated release rate as a function of time. (D) Peak thrombin-mediated release rate as a function of thrombin concentration. Table S1. Summary of results from thrombin map by LC-MS (TOF) analysis. For SC rFVIIIFc R1645A/R1648A, mass analysis confirmed the presence of the R1645A/R1648A mutations. (DOCX)