title: Separation of human immunoglobulin G subclasses on a protein A monolith column.
abstract: Monolithic columns have attracted significant attention for the purification of large biomolecules. In the present study, a step gradient elution method was evaluated for the separation of human immunoglobulin G (hIgG) into its subclasses on CIM (convective interaction media) r-protein A (recombinant protein A) monolithic column. hIgG was loaded onto the column and bound protein was eluted with a pH gradient. The subclass content of the eluted fractions was analyzed by enzyme-linked immunosorbent assay (ELISA). Results showed that separation of IgG3 from the other three subclasses can be successfully achieved with high selectivity (100%) and throughput on monolithic media. It was also revealed that enriched fractions of IgG1 and IgG2 could be obtained from purified hIgG in a 28min long chromatographic run. Three fractions with high IgG1 content (89.1%, 94.3% and 88.8%) were recovered. Furthermore, IgG2 was enriched to 64% successfully. A rapid step gradient elution scheme without any additives in buffers was proven to obtain enriched preparations of the two important subclasses with high throughput. The separation time can be reduced even more by increasing the flow rate without any loss in selectivity, which will be beneficial in industrial scale applications.
authors.affiliation: Laboratory of Separation and Reaction Engineering, Associate Laboratory LSRE/LCM, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal. Electronic address: pellin.arslan@gmail.com.
authors.forename: Pelin
authors.initials: P
authors.lastname: Leblebici
authors.affiliation: Laboratory of Separation and Reaction Engineering, Associate Laboratory LSRE/LCM, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal. Electronic address: enisleblebici@gmail.com.
authors.forename: M Enis
authors.initials: ME
authors.lastname: Leblebici
authors.affiliation: IBMC-Institute for Molecular and Cell Biology, University of Porto, 4150-180 Porto, Portugal. Electronic address: ffsilva@ibmc.up.pt.
authors.forename: Frederico
authors.initials: F
authors.lastname: Ferreira-da-Silva
authors.affiliation: Laboratory of Separation and Reaction Engineering, Associate Laboratory LSRE/LCM, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal. Electronic address: arodrig@fe.up.pt.
authors.forename: Alírio E
authors.initials: AE
authors.lastname: Rodrigues
authors.affiliation: Laboratory of Separation and Reaction Engineering, Associate Laboratory LSRE/LCM, School of Technology and Management, Polytechnic Institute of Bragança, Campus de Santa Apolónia, Apartado 1134, 5301-857 Bragança, Portugal. Electronic address: pais@ipb.pt.
authors.forename: Luís S
authors.initials: LS
authors.lastname: Pais
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis / Q000145:classification / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry; D013205:Staphylococcal Protein A / Q000737:chemistry*
chemical_list: D007074:Immunoglobulin G; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A
keywords: Immunoglobulin G subclasses; Monolith; Protein A chromatography; Separation