Abstract
Antigen binding fragments (Fabs) used in research (e.g., antibody mimetics, antibody-drug conjugate, bispecific antibodies) are frequently obtained by enzymatic digestion of monoclonal antibodies using immobilised papain. Despite obtaining pure Fab, using immobilised papain to digest IgG has limitations, most notably slow digestion time (more than 8 h), high cost and limited scalability. Here we report a time and cost-effective method to produce pure, active and stable Fab using soluble papain. Large laboratory scale digestion of an antibody (100 mg) was achieved using soluble papain with a digestion time of 30 min and isolated yields of 55-60%. The obtained Fabs displayed similar binding activity as Fabs prepared via immobilised papain digestion. Site-specific conjugation between Fabs and polyethylene glycol (PEG) was carried out to obtain antibody mimetics FpF (Fab-PEG-Fab) indicating that the native disulphide bond had been preserved. Surface-plasmon resonance (SPR) of prepared FpFs showed that binding activity towards the intended antigen was maintained. We anticipate that this work will provide a fast and less costly method for researchers to produce antibody fragments at large scale from whole IgG suitable for use in research.