Engineering and production of HB-002.1. (A) Diagram of HB-002.1 engineered structure. Illustration on top represents Flt1-Fc consisting of the D2 domain only fused with the Fc portion of human IgG1. HB-002.1 contains the D2 domain plus 5 and 2 amino acids at the 5' and 3' flanking region respectively. Signal peptide derived from the heavy chain of mouse IgG1 (LS) was included for both constructs. (B) SDS-PAGE gel analysis. Three proteins were included in the analysis: HB-002.1 (Lane 1, 4); Bevacizumab (Lane 2, 5); Flt1-Fc (Lane 3, 6). 5 μg of each protein were loaded under reducing and non-reducing conditions. (C-D), Western blot analysis. 0.25, 0.5, and 1 μg of HB-002.1 protein and 1 μg of hIgG-Fc were resolved on 10% SDS-PAGE gels under reducing (R) or non-reducing (NR) conditions, transferred to a polyvinylidene difluoride membrane, and probed with Fc-specific (C) or VEGFR1-specific (D) antibody (Ab). For comparison, protein MW size markers are shown in kDa.