Extractive fractionation of equine hyperimmune plasma

Aqueous two-phase partition and triphasic systems containing thiophilic and metal ligands were used to produce anti-snake venom serum using the fracti onation of hyperimmune equine plasma with neutralizing activity against Bot hrops spp. venom as the model. In poly (ethyleneglycol) (PEG)/salt systems, PEG molecular weight, system p H, and the amount of polyvinylpirrolidone (PVP) added were optimized in ord er to promote selective immunoglobulin recovery. From these studies a PEG 1 500/ammonium sulfate system, pH 8.0, a tie-line length parameter (TLL) 21%, and a 2% PVP K-12 were preliminarily selected. The immunoreactive fraction (ELISA) mainly preferred the top phase; with a theoretical yield of > 97% and a purification factor of 3.5. Practical recoveries were, however, hampe red by limited product solubility and an erratic global process performance . In order to overcome these limitations, we moved to a system formed by deri vatives of cellulose and starch, which contained an affinity solid phase. T his three-phase system was compatible with the use of thiophilic or immobil ized metal ion affinity (IMAC) supports, the first showing the higher selec tivity. The system composed of 2% Methocel, 2% Reppal, 1.0 mol/kg ammonium sulfate, and 5-10% 2S-thioether sulfone agarose allowed recovery of 52% of the immunoreactive fraction from hyperimmune equine plasma at a total prote in concentration of 0.25%. This process strategy also allowed simple operat ion and easy of affinity ligand recycling.