Separation of human immunoglobulin G subclasses on a protein A monolith column

• Type: Article in International Scientific Journal
• Date: 2014

Authors

Pelin Leblebici (Author) Other; M. Enis Leblebici (Author) Other; Frederico Ferreira da Silva (Author) Other; Alírio E. Rodrigues (Author) FEUP; Luís S. Pais (Author) Other

Journal

Title: Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences

Vol. 962

Pages: 89-93

ISSN: 1570-0232

Publisher: Elsevier

Indexing

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Associated Projects

LA 20 - PROJECTO ESTRATÉGICO - LA 20 - 2013-2014

Other information

Authenticus ID: P-009-HCF

DOI: 10.1016/j.jchromb.2014.05.029

Abstract (EN): 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) Gamma-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 28 min 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.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 5