Development of Analytical Methods for Characterization of Fab Fragments

Abstract

The constant need for increased sensitivity of antibody‐based assays has increased the need for high affinity molecules. Use of Fab antibody fragments can increase the specificity and sensitivity of immunoassays. We developed simple GPC, SDS‐PAGE and IEF methods that can be used to characterize Fab material produced by reduction of F(ab’)2 fragments. Specific reduction of F(ab’)2 to Fab was accomplished using cysteamine HCl. The resulting Fab fragments were analyzed with and without alkylation with iodoacetamide. Fab fragments were analyzed by gel permeation chromatography using a Waters HPLC system and multiple column types. SDS‐PAGE was performed using multiple gel types and running conditions. Scanning densitometry was performed using a Bio‐Rad GS800 and electrophoretic profiles were determined. The pI range and IEF profiles of the Fab samples were determined using a Phastsystem (GE Healthcare). The methods developed provide reproducible profiles for both relative reduced fragment content and identity. The methods were capable of detecting low percentage impurities. These methods provide quick analytical protocols that can be used to characterize Fab fragments for use in immunoassays.