Protein Expression and Purification, Vol.41, No.1, 68-76, 2005
Purification and characterization of a single-chain chimeric anti-p185 antibody expressed by CHO-GS system
Monoclonal antibody A21 reacts specifically with the extracellular domain of p185(c-erbB-2) oncoprotein, a member of the epidermal growth factor receptor family. In a previous study, we constructed a single-chain chimeric antibody, assembled using an A21 single-chain Fv antibody and a human IgG1 Fc fragment. In this study, we expressed this chimeric antibody using a CHO-GS system, and developed a simple and efficient method for its purification. After only one step using affinity purification, the recovery rate and purity of the antibody attained was 60 and 91 %, respectively. After a second step, using reverse phase HPLC purification, the purity was above 99 %. The high purity of the recombinant antibody allowed us to identify a number of its intrinsic molecular properties, including antigen binding activity, measurement of affinity constant, N-terminal sequencing, and mass spectrometer analysis. These results further augment the potential of this recombinant antibody to be a drug candidate for cancer therapy. (c) 2004 Elsevier Inc. All rights reserved.