title: Isolation and purification of blood group antigens using immuno-affinity chromatography on short monolithic columns.
abstract: 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(®)) 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(®) Disk with epoxy chemistry. After this, the immobilized CIM(®) 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(®) Tubes. In comparison to conventional affinity chromatography, blood group antigens were also purified via His-tag using a HiTrap(®) 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.
authors.affiliation: Institute for Technical Chemistry, Gottfried Wilhelm Leibniz Universität Hannover, Callinstraße 5, D-30167 Hanover, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Mönster
authors.forename: Oliver
authors.initials: O
authors.lastname: Hiller
authors.forename: Daniela
authors.initials: D
authors.lastname: Grüger
authors.forename: Rainer
authors.initials: R
authors.lastname: Blasczyk
authors.forename: Cornelia
authors.initials: C
authors.lastname: Kasper
mesh_terms: D055910:Antibodies, Immobilized / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001789:Blood Group Antigens / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D055910:Antibodies, Immobilized; D000911:Antibodies, Monoclonal; D001789:Blood Group Antigens; D003470:Culture Media; D011993:Recombinant Fusion Proteins