title: Light extinction and scattering by agarose based resin beads and applications in high-throughput screening.
abstract: Optimization of chromatographic processes by high-throughput screening (HTS) methodologies have become a critical part of downstream process development. Nevertheless there are still no non-invasive optical methods to characterize resin as well as protein-resin interaction on liquid-handling platforms available. Several approaches to automated resin screening in microplates are described in literature, yet all those methods involve indirect measurements by removal of, and sample quantification within, supernatant volumes. In this work, we introduce light extinction by light scattering to directly assess resin volume and bead density within microplates. Methods for this novel resin characterization are described for 96 and 384-well microplates. An example application demonstrates ligand concentration measurement in microplates with four commercial SP Sepharose™ Fast Flow batches. Further, direct quantification of adsorbent bound biomolecules is shown in an example with kinetic protein-resin interaction measurement in a batch screening process. This new approach is expected to promote batch-based resin characterization and monitoring on HTS platforms and further miniaturization and increase in throughput of chromatographic HTS processes.
authors.affiliation: Section IV: Biomolecular Separation Engineering, Institute of Engineering in Life Sciences, Karlsruhe Institute of Technology (KIT), Engler-Bunte-Ring 1, 76131 Karlsruhe, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Kittelmann
authors.affiliation: Section IV: Biomolecular Separation Engineering, Institute of Engineering in Life Sciences, Karlsruhe Institute of Technology (KIT), Engler-Bunte-Ring 1, 76131 Karlsruhe, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Hämmerling
authors.affiliation: Section IV: Biomolecular Separation Engineering, Institute of Engineering in Life Sciences, Karlsruhe Institute of Technology (KIT), Engler-Bunte-Ring 1, 76131 Karlsruhe, Germany.
authors.forename: Moritz
authors.initials: M
authors.lastname: Ebeler
authors.affiliation: Section IV: Biomolecular Separation Engineering, Institute of Engineering in Life Sciences, Karlsruhe Institute of Technology (KIT), Engler-Bunte-Ring 1, 76131 Karlsruhe, Germany. Electronic address: Juergen.Hubbuch@kit.edu.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Hubbuch
mesh_terms: D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002845:Chromatography / Q000379:methods*; D057166:High-Throughput Screening Assays; D007700:Kinetics; D008024:Ligands; D008027:Light; D011506:Proteins / Q000032:analysis; D012685:Sepharose / Q000737:chemistry*
chemical_list: D008024:Ligands; D011506:Proteins; D012685:Sepharose
keywords: Batch chromatography; High-throughput screening (HTS); Ion exchange; Ionic capacity; Light scattering; Resin quantification