Isoelectric Points Determination
The isoelectric points were measured by capillary isoelectric focusing (cIEF) for each BsAb using Protein Simple iCE3 with autosampler on a FC-coated cIEF Cartridge (Protein Simple Part # 101701). The samples were prepared at 0.25mg/ml in 3M urea/0.35% methylcellulose with Pharmalytes 3-10 (Sigma-Aldrich Cat.# 17-0456-01) and pI markers (ProteinSimple). Samples were focused at 1500V for 1-minute followed by a second focusing period of 3000V for 8-minutes. Antibody isoelectric points were calibrated to low and high internal pI makers. The pI of each the Fab and scFv was calculated from the primary sequences using algorithm in Molecular Operating Environment (MOE2019) (Chemical Computing Group, Montreal, Canada) and outlined by Sillero and colleagues.31