Reformatting and Purification of Chimeric Antibodies

The reformatting, expression, and purification of full-length antibodies was performed as described previously [31 (link),35 (link)]. Isolated yeast vectors were sequenced, and the VL fragment was reformatted into pTT5-derived vectors by golden gate assembly. For the soluble expression of full-length chimeric antibodies, Expi293F^TM cells (Thermo Fisher Scientific) were transiently transfected using the ExpiFectamine^TM 293 Transfection Kit (Thermo Fisher Scientific) following the manufacturer’s protocol. For purification, cell culture supernatants were collected five days post transfection, sterile-filtered, and applied to a MabSelect^TM PrismA HP column (GE Healthcare, Piscataway, NJ, USA) using an ÄKTA pure^TM chromatography system (GE Healthcare). One-armed molecules were captured by IMAC (HisTrap HP, GE Healthcare), followed by Strep-Tactin XT affinity chromatography according to the manufacturer’s protocol. Buffer exchange against PBS was performed using a HiTrap^TM Desalting column (GE Healthcare).