Format

Send to

Choose Destination
See comment in PubMed Commons below
J Proteomics. 2010 Jan 3;73(3):678-88. doi: 10.1016/j.jprot.2009.09.020. Epub 2009 Oct 9.

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

Author information

1
Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, RI, USA.

Abstract

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.

PMID:
19819359
PMCID:
PMC2818390
DOI:
10.1016/j.jprot.2009.09.020
[Indexed for MEDLINE]
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments

    Supplemental Content

    Full text links

    Icon for Elsevier Science Icon for PubMed Central
    Loading ...
    Support Center