Studies on the purification of antibody fragments

Abstract Antigen-binding fragments (Fabs) are becoming increasingly prevalent as an alternative to full-length monoclonal antibodies. In this work, the digestion of a mixture of different human antibodies isotypes was optimized in terms of different parameters, including the concentration of papain and cysteine, and the digestion time. The recovery of the Fab fragments was subsequently evaluated by designing four different downstream purification schemes, where the use of affinity chromatography (protein A and L) was the most efficient to isolate the Fab fragments after IgG digestion. A rapid screening of optimal conditions for the binding of pure Fab fragments to four non-affinity chromatography resins was then performed using micro-columns fabricated in a PDMS microfluidic device. The goal of these studies was to screen and evaluate the performance of two cation exchange (carboxymethyl and heparin) and two multimodal (Capto MMC and phenyl boronate) ligands for the capture of Fabs under a wide range of pH (5–9) and conductivity (up to 8 mS/cm) conditions. Multimodal resins showed the best results in binding Fab fragments, particularly at pH 5, well below the range of isoelectric points of the target Fab molecules. In addition, these resins demonstrated to have a salt-tolerant behaviour, meaning that the binding of Fab fragments was not significantly impacted when the conductivity of the adsorption buffer was increased to near-physiological conditions (8 mS/cm).