Naslov
Use of proteomics for validation of the isolation process of clotting factor IX from human plasma

Autori
Clifton, James ; Huang, Feilei ; Gašo-Sokač, Dajana ; Brilliant, Kate ; Hixson, Douglas ; Josić, Djuro

Izvornik
Journal of proteomics (1874-3919) 73 (2010), 3; 678-688

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
Clotting factor IX; vitamin K dependent clotting factors; proteomics; human plasma; fractionation

Sažetak
The use of proteomic techniques in the monitoring of different production steps of plasma-derived clotting factor IX(pd F IX)was demonstrated. The first step, solid-phase extractionwith a weak anion-exchange resin, fractionates the bulk of human serum albumin (HSA), immunoglobulin G, and other non-binding proteins from F IX. The proteins that strongly bind to the anion-exchange resin are eluted by higher salt concentrations. In the second step, anion-exchange chromatography, residual HSA, some proteases and other contaminating proteins are separated. In the last chromatographic step, affinity chromatography with immobilized heparin, the majority of the residual impurities are removed. However, some contaminating proteins still remain in the eluate fromthe affinity column. The next step in the production process, virus filtration, is also an efficient step for the removal of residual impurities, mainly high molecular weight proteins, such as vitronectin and inter-alpha inhibitor proteins. In each production step, the active component, pd F IX and contaminating proteins aremonitored by biochemical and immunochemicalmethods and by LC–MS/MS and their removal documented. Ourmethodology is very helpful for further process optimization, rapid identification of target proteins with relatively low abundance, and for the design of subsequent steps for their removal or purification.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija

POVEZANOST RADA