Isolation and purification of blood group antigens using immuno-affinity chromatography on short monolithic columns

Monolithic columns have gained increasing attention as stationary phases for the separation of biomolecules and biopharmaceuticals. In the present work the performance of monolithic convective interaction media (CIM (R)) chromatography for the purification of blood group antigens was established. The proteins employed in this study are derived from blood group antigens Knops. JMH and Scianna, equipped both with a His-tag and with a V5-tag by which they can be purified. In a first step a monoclonal antibody directed against the V5-tag was immobilized on a CIM (R) Disk with epoxy chemistry. After this, the immobilized CIM (R) Disk was used in immuno-affinity chromatography to purify the three blood group antigens from cell culture supernatant. Up-scaling of the applied technology was carried out using CIM (R) Tubes. In comparison to conventional affinity chromatography, blood group antigens were also purified via His-tag using a HiTrap (R) metal-affinity column. The two purifications have been compared regarding purity, yield and purification speed. Using the monolithic support, it was possible to isolate the blood group antigens with a higher flow rate than using the conventional bed-packed column. (C) 2010 Elsevier B.V. All rights reserved.