Browsing by Author "Ferreira-da-Silva, Frederico"
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- Separation of human immunoglobulin G subclasses on a protein A monolith columnPublication . Leblebici, Pelin; Leblebici, Mumin Enis; Ferreira-da-Silva, Frederico; Rodrigues, Alírio; Pais, Luís S.Monolithic columns have attracted significant attention for the purification of large biomolecules. In thepresent study, a step gradient elution method was evaluated for the separation of human immunoglobulinG (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 gra-dient. The subclass content of the eluted fractions was analyzed by enzyme-linked immunosorbentassay (ELISA). Results showed that separation of IgG3 from the other three subclasses can be success-fully achieved with high selectivity (100%) and throughput on monolithic media. It was also revealedthat enriched fractions of IgG1 and IgG2 could be obtained from purified hIgG in a 28 min long chro-matographic 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 anyadditives in buffers was proven to obtain enriched preparations of the two important subclasses withhigh throughput. The separation time can be reduced even more by increasing the flow rate without anyloss in selectivity, which will be beneficial in industrial scale applications.
- Separation of human immunoglobulin G subclasses using monolith technologyPublication . Leblebici, Pelin; Leblebici, Mumin Enis; Ferreira-da-Silva, Frederico; Ribeiro, António E.; Rodrigues, Alírio; Pais, Luís S.In the present study, a step gradient elution method was evaluated for the separation of human immunoglobulin G (hIgG) into its subclasses on CIM rwprotein A monolithic column. hIgG Seheme 1. Represantative structures of the human IgO subclasses [I). was loaded onto the column and bound protein was eluted with a pH gradient. The subclass content of the eluted fractions was analyzed by enzymewIinked 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 IgG 1 and IgG2 could be obtained from purified hIgG in a 28 minutes long chromatographic run, Three fractions with high IgO 1 content (89.1%,94,3% and 88.8%) were recovered, Furthennore, IgG2 was enriched to 64% successfully. A rapid step gradient elution scheme without any additives in buffers was proven to obtain enriched prep~ions of the two important subclasses with high througbput.