title: Improving the binding capacities of protein A chromatographic materials by means of ligand polymerization.
abstract: Protein A chromatography is one of the most important techniques used in the purification of monoclonal antibodies. Due to the low dynamic binding capacity of protein A chromatographic materials compared to other stationary phases, protein A chromatography is often discussed to be the bottleneck among current purification processes. Several approaches were tested within this study in order to maximize IgG binding capacities of current acrylamido-based based resins. Genetic engineering techniques were used in order to polymerize one of the IgG binding domains (B-domain) of protein A from Staphylococcus aureus (SpA) to achieve ligands with an increased length. The solution-binding ratio and the total size of ligand-antibody complexes were used to characterize the interaction potential of novel ligands, revealing a relatively linear dependency between the number of binding domains upon the amount of bound antibody molecules. This relationship was also valid up to a ligand which was comprised of 8 B-domains after attaching them onto acrylamido-based based stationary phases using epoxy coupling techniques. Equilibrium binding capacities of more than 80mghIgGmL(-1) were achieved using the B8 ligand. Furthermore, static binding capacities, especially for smaller ligands comprised of fewer B-domains, were improved up to 87mghIgGmL(-1) using site-specific coupling chemistry, which is an improvement of more than 20% compared to commercially available materials. In order to evaluate pore exclusion effects due to the use of prolonged affinity ligands, prepared materials were characterized regarding their effective intraparticle porosity and breakthrough capacity.
authors.affiliation: Faculty for Mechanical Engineering, Bioseparation Engineering Group, Technische Universität München, Boltzmannstr. 15, 85748 Garching, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Freiherr von Roman
authors.affiliation: Faculty for Mechanical Engineering, Bioseparation Engineering Group, Technische Universität München, Boltzmannstr. 15, 85748 Garching, Germany. Electronic address: s.berensmeier@tum.de.
authors.forename: Sonja
authors.initials: S
authors.lastname: Berensmeier
mesh_terms: D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D005818:Genetic Engineering; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics; D008024:Ligands; D058105:Polymerization; D011485:Protein Binding; D013205:Staphylococcal Protein A / Q000737:chemistry* / Q000235:genetics
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D008024:Ligands; D013205:Staphylococcal Protein A
keywords: Improved binding capacity; Ligand development; Protein A chromatography; Site-specific immobilization