Effective flow-through polishing strategies for knob-into-hole bispecific antibodies

Abstract Bispecific antibodies (bsAbs), though possessing great therapeutic potential, are extremely challenging to obtain at high purity within a limited number of scalable downstream processing steps. Complementary Protein A chromatography, polishing strategies play critical role removing the remaining molecular weight (HMW) and low (LMW) species, as well host cell proteins (HCP) in order achieve final product purity. Here, we demonstrate using two knob-into-hole (KiH) bsAb constructs that flow-through steps utilising Capto Butyl ImpRes adhere resins, performed after an optimal affinity chromatography step can further reduce HCP by 17- 35-fold HMW LMW species with respect monomer ~ 4–6% 1%, respectively, meet therapeutical requirement 30–60 mg/mL-resin (R) load. This complete strategy, guided Design Experiments (DoE), eliminates undesirable aggregation problems associated higher propensity scFv containing bsAbs may occur bind elute mode, offering improved ease overall process operation without additional elution buffer preparation consumption, thus aligning intensification efforts. Overall, through employment (1) (2) steps, < 1% 100 ppm be obtained recovery 56–87%. Graphical