Preparation and high resolution analysis of a Fab fragment

Abstract

In this work a Fab fragment was produced by papain cleavage of a purified monoclonal antibody (Mab) followed by purification on MabSelect™ Sure™ and Capto™ L media (protein A and protein L, respectively, immobilized onto high flow agarose). Fractions from the different steps were analyzed by gel filtration (GF) to follow purification progress. The process started with Mab being purified from feed on MabSelect Sure and thereafter the Mab was digested by papain overnight at 37 degrees into Fab‐ and Fcfragments. Cleavage was interrupted with the inhibitor antipain and the digest was applied to a column containing MabSelect Sure. Fc and partially digested Mab bind to the chromatography medium whereas the Fab fragment elutes in the flow through together with other contaminants such as papain. The flow through fraction was applied to Capto L medium and the Fab fragment was thus further purified. The purity of the Fab fragment was confirmed by analysis on new high resolution GF media. This new GF media has the resolution power to separate both Mab from aggregates and Fab fragments from Fc fragments.