VB6-845 is a recombinant Fab-format cytotoxin that is currently being evaluated in a phase I clinical trial. It is expressed in E. coli using a dicistronic expression unit: the first unit is comprised of the VH-CH domain containing a histidine affinity tag placed at the N-terminus, and the second unit is comprised of a cytotoxic bouganin protein linked C-terminally to the VL-CL domain via a furin-sensitive linker. The dicistronic unit is cloned into the pING3302 expression vector under the control of the arabinose–inducible araBAD promoter. Both the VH-CH and VL-CH-cytotoxin expression units are preceded by a PelB leader sequence that targets both polypeptide chains into the periplasmic space, and allows the recovery of fully formed and active VB6-845 in the culture supernatant using a six-step purification process. Optimization of fermentation parameters and the VB6-845 coding region yielded ~100-fold increase in titers to 100 mg/L at both the 15 L development scale to the 1,200 L production scale. Furthermore, implementation of improved harvesting conditions significantly ameliorated overall product recovery