title: Investigation of protein selectivity in multimodal chromatography using in silico designed Fab fragment variants.
abstract: In this study, a unique set of antibody Fab fragments was designed in silico and produced to examine the relationship between protein surface properties and selectivity in multimodal chromatographic systems. We hypothesized that multimodal ligands containing both hydrophobic and charged moieties would interact strongly with protein surface regions where charged groups and hydrophobic patches were in close spatial proximity. Protein surface property characterization tools were employed to identify the potential multimodal ligand binding regions on the Fab fragment of a humanized antibody and to evaluate the impact of mutations on surface charge and hydrophobicity. Twenty Fab variants were generated by site-directed mutagenesis, recombinant expression, and affinity purification. Column gradient experiments were carried out with the Fab variants in multimodal, cation-exchange, and hydrophobic interaction chromatographic systems. The results clearly indicated that selectivity in the multimodal system was different from the other chromatographic modes examined. Column retention data for the reduced charge Fab variants identified a binding site comprising light chain CDR1 as the main electrostatic interaction site for the multimodal and cation-exchange ligands. Furthermore, the multimodal ligand binding was enhanced by additional hydrophobic contributions as evident from the results obtained with hydrophobic Fab variants. The use of in silico protein surface property analyses combined with molecular biology techniques, protein expression, and chromatographic evaluations represents a previously undescribed and powerful approach for investigating multimodal selectivity with complex biomolecules.
authors.affiliation: Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, 12180.|Protein Purification Technology, Novo Nordisk A/S, DK-2760 Maaloev, Denmark.
authors.forename: Hanne Sophie
authors.initials: HS
authors.lastname: Karkov
authors.affiliation: Yeast Expression Systems, Novo Nordisk A/S, DK-2760 Maaloev, Denmark.
authors.forename: Berit Olsen
authors.initials: BO
authors.lastname: Krogh
authors.affiliation: Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, 12180.
authors.forename: James
authors.initials: J
authors.lastname: Woo
authors.affiliation: Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, 12180.
authors.forename: Siddharth
authors.initials: S
authors.lastname: Parimal
authors.affiliation: Protein Purification Technology, Novo Nordisk A/S, DK-2760 Maaloev, Denmark.
authors.forename: Haleh
authors.initials: H
authors.lastname: Ahmadian
authors.affiliation: Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, 12180. crames@rpi.edu.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Cramer
mesh_terms: D001709:Biotechnology / Q000379:methods; D002846:Chromatography, Affinity / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D019295:Computational Biology; D004252:DNA Mutational Analysis; D057927:Hydrophobic and Hydrophilic Interactions; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D013678:Technology, Pharmaceutical / Q000379:methods
chemical_list: D007140:Immunoglobulin Fab Fragments
keywords: Fab fragment variants; in silico design; multimodal chromatography; protein binding orientations; protein separations; protein surface properties