Optimization of metal dependent antibodies for chromatography

Abstract

This study focuses on the utilization of metal-dependent monoclonal antibodies for large-scale chromatography and addresses an aspect that has been cited to lower immunosorbent performance, namely "orientation" of antibodies on matrices. The antibodies used in this study, the "EDTAdependent" 7D7BlO and the "Ca2+ -dependent" HPC4 are directed against human Protein C (PC).

The 7D7BI0 antibody was characterized in terms of its metaldependency and specificity. The region of PC (epitope) recognized by 7D7BlO was identified as the first 15 residues in the NH₂-terminal. Immunosorbents made with 7D7BI0 provided highly pure and functional PC.

The "orientation" of the antibodies on matrices was addressed in two ways. In the first approach, performance of immunosorbents coupled through carbohydrate moieties were compared with immunosorbents coupled through peptide regions. Coupling via carbohydrate linkages, which is generally believed to be Fc-directed, did not have any advantage in terms of efficiency and recovery over coupling via peptide.