A novel core fractionation process of human plasma by expanded bed adsorption chromatography.
Current plasma fractionation technology combines ethanol precipitation with packed bed chromatography. We have developed a novel core fractionation process comprising five expanded bed adsorption (EBA) chromatographic steps on high-density modified agarose/tungsten carbide beads. Plasma was first chromatographed on two diethyl amino-ethyl (DEAE)-tungsten carbide agarose adsorbents (respective mean particle diameters of d(v)(0.5)=190 and 37 microm) to isolate at 50 to 80% recovery a fraction containing 4 to 7 IU/ml factor II (FII), factor IX (FIX), and factor X (FX) (specific activity >1 IU/mg) and another enriched in FVIII and von Willebrand factor (vWF) (approximately 1 IU/ml and 0.6 IU/mg, respectively). The flow-through was adsorbed on 4% agarose-10% tungsten carbide beads coupled with an acidic mixed-mode ligand to isolate an 80% pure immunoglobulin G (IgG) at a 93% step recovery. A highly purified alpha1-antitrypsin was isolated at 95% step recovery by adsorbing the flow-through on 4% epoxy-crosslinked agarose-10% tungsten carbide adsorbent material coupled with a cationic ligand. Isolation of 98% pure albumin was achieved at a 99% step recovery by pH 4.5 adsorption of the flow-through on 6% agarose-10% tungsten carbide beads coupled with an acidic mixed-mode ligand. EBA may represent a feasible alternative core plasma fractionation tool.
Lihme
Allan
A
Upfront Chromatography, 2100 Copenhagen, Denmark. al@upfront-dk.com
Hansen
Marie Bendix
MB
Andersen
Inga Vaarst
IV
Burnouf
Thierry
T
D001779:Blood Coagulation Factors / Q000302:isolation & purification; D001798:Blood Proteins / Q000302:isolation & purification*; D005591:Chemical Fractionation; D002850:Chromatography, Gel / Q000379:methods*; D000431:Ethanol / Q000737:chemistry; D005169:Factor VIII / Q000302:isolation & purification; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification; D012709:Serum Albumin / Q000302:isolation & purification; D017973:Tungsten Compounds / Q000737:chemistry; D000515:alpha 1-Antitrypsin / Q000302:isolation & purification; D014841:von Willebrand Factor / Q000302:isolation & purification
D001779:Blood Coagulation Factors; D001798:Blood Proteins; D007074:Immunoglobulin G; D012709:Serum Albumin; D017973:Tungsten Compounds; D000515:alpha 1-Antitrypsin; D014841:von Willebrand Factor; C002802:tungsten carbide; C025667:prothrombin complex concentrates; D000431:Ethanol; D005169:Factor VIII