Use of proteomics for validation of the isolation process of clotting factor IX from human plasma.
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 extraction with 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 from the 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 are monitored by biochemical and immunochemical methods and by LC-MS/MS and their removal documented. Our methodology 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.
Clifton
James
J
Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, RI, USA.
Huang
Feilei
F
Gaso-Sokac
Dajana
D
Brilliant
Kate
K
Hixson
Douglas
D
Josic
Djuro
D
D000837:Anion Exchange Resins / Q000737:chemistry / Q000494:pharmacology; D001779:Blood Coagulation Factors / Q000032:analysis / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods; D002847:Chromatography, Agarose / Q000379:methods; D019411:Clinical Laboratory Techniques; D003637:DEAE-Dextran / Q000737:chemistry / Q000494:pharmacology; D005164:Factor IX / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D006440:Hemofiltration / Q000379:methods; D006493:Heparin / Q000378:metabolism; D006801:Humans; D010949:Plasma / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D052616:Solid Phase Extraction / Q000379:methods; D054928:Validation Studies as Topic
D000837:Anion Exchange Resins; D001779:Blood Coagulation Factors; D005164:Factor IX; D006493:Heparin; D003637:DEAE-Dextran