Figure 1.
Development of BIVV001. (A) Construct 1: rFVIIIFc-VWF(DʹD3). rFVIIIFc is stabilized by covalently attaching a D1D2DʹD3 (C1099A/C1142A) domain of VWF through immunoglobulin-G1 Fc molecules, which prevents interaction of rFVIIIFc with endogenous full-length VWF. A R1648A mutation in FVIII prevents FVIII processing into heavy and light chains. Construct 2: rFVIIIFc-VWF-XTEN. One 288-amino-acid XTEN polypeptide is inserted into the B-domain region of FVIII, a second 144-amino-acid XTEN polypeptide is inserted between DʹD3 and Fc. Construct 3: rFVIIIFc-VWF-XTEN. The LVPR thrombin site located between DʹD3 and Fc on constructs 1 and 2 was replaced with an FVIII acidic region 2 (a2) thrombin site. Construct 4: rFVIIIFc-VWF-XTEN (BIVV001). Three amino acid residues (GAP) from the FVIII/XTEN junction and 9 amino acids (PPVLKRHQA) from the FVIII B-domain linker were removed to avoid potential MHCII-binding sites. (B) BIVV001 is generated by coexpressing 2 polypeptide chains in HEK293 cells, a human cell line. Purified component parts of BIVV001 were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; BioRad stain-free gel; 4%-20%) under reduced (R) and nonreducing (NR) conditions. See supplemental Methods for more details. FVIII-XTEN-Fc portion is expressed in the first chain (band F8; NR and R lane) and DʹD3-XTEN-Fc portion (band VD; R lane) is expressed in the second chain. The 2 chains are held together by disulfide bonds in the Fc region (heterodimer band [HD]; NR lane). The D1D2 (propeptide) is removed during intracellular processing. Size-exclusion chromatography (SEC) was performed using high-performance liquid chromatography (1260 Bioinert) with a BEH450 column and run isocratically at 0.3 mL/min for 18 minutes. (See supplemental Methods for more details.) The SEC profile of BIVV001 under nonreducing conditions is shown; the predicted molecular mass of BIVV001 is ∼312 kDa, but, because of the presence of XTEN, it has a large hydrodynamic radius and elutes before 670 kDa. MW, molecular weight; PACE, paired basic amino acid cleaving enzyme; Tyr, tyrosine.

Development of BIVV001. (A) Construct 1: rFVIIIFc-VWF(DʹD3). rFVIIIFc is stabilized by covalently attaching a D1D2DʹD3 (C1099A/C1142A) domain of VWF through immunoglobulin-G1 Fc molecules, which prevents interaction of rFVIIIFc with endogenous full-length VWF. A R1648A mutation in FVIII prevents FVIII processing into heavy and light chains. Construct 2: rFVIIIFc-VWF-XTEN. One 288-amino-acid XTEN polypeptide is inserted into the B-domain region of FVIII, a second 144-amino-acid XTEN polypeptide is inserted between DʹD3 and Fc. Construct 3: rFVIIIFc-VWF-XTEN. The LVPR thrombin site located between DʹD3 and Fc on constructs 1 and 2 was replaced with an FVIII acidic region 2 (a2) thrombin site. Construct 4: rFVIIIFc-VWF-XTEN (BIVV001). Three amino acid residues (GAP) from the FVIII/XTEN junction and 9 amino acids (PPVLKRHQA) from the FVIII B-domain linker were removed to avoid potential MHCII-binding sites. (B) BIVV001 is generated by coexpressing 2 polypeptide chains in HEK293 cells, a human cell line. Purified component parts of BIVV001 were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; BioRad stain-free gel; 4%-20%) under reduced (R) and nonreducing (NR) conditions. See supplemental Methods for more details. FVIII-XTEN-Fc portion is expressed in the first chain (band F8; NR and R lane) and DʹD3-XTEN-Fc portion (band VD; R lane) is expressed in the second chain. The 2 chains are held together by disulfide bonds in the Fc region (heterodimer band [HD]; NR lane). The D1D2 (propeptide) is removed during intracellular processing. Size-exclusion chromatography (SEC) was performed using high-performance liquid chromatography (1260 Bioinert) with a BEH450 column and run isocratically at 0.3 mL/min for 18 minutes. (See supplemental Methods for more details.) The SEC profile of BIVV001 under nonreducing conditions is shown; the predicted molecular mass of BIVV001 is ∼312 kDa, but, because of the presence of XTEN, it has a large hydrodynamic radius and elutes before 670 kDa. MW, molecular weight; PACE, paired basic amino acid cleaving enzyme; Tyr, tyrosine.

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