CAMPERI Silvia Andrea
Extractive fractionation of equine hyperimmune plasma
GUSTAVO H. KIJAK; SILVIA A. CAMPERI; RITA STUMPO; OSVALDO CASCONE; HECTOR M. FERNÁNDEZ LAHORE
SEPARATION SCIENCE AND TECHNOLOGY
Taylor & Francis Group
Año: 2001 vol. 36 p. 59 - 59
Aqueous two-phase partition and triphasic systems containing thiophilic and metal ligands were used to produce antisnake venom serum using the fractionation of hyperimmune equine plasma with neutralizing activity against Bothrops spp. venom as the model. In poly(ethyleneglycol)(PEG)/salt systems, PEG molecular weight, system pH, and the amount of polyvinylpirrolidone (PVP) added were optimized in order to promote selective immunoglobulin recovery. From these studies a PEG 1500/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, hampered by limited product solubility and an erratic global process performance. In order to overcome these limitations, we moved to a system formed by derivatives of cellulose and starch, which contained an affinity solid phase. This three-phase system was compatible with the use of thiophilic or immobilized metal ion affinity (IMAC) supports, the first showing the higher selectivity. The system composed of 2% Methocel, 2% Reppal, 1.0 mol/kg ammonium sulfate, and 510% 2S-thioether sulfone agarose allowed recovery of 52% of the immunoreactive fraction from hyperimmune equine plasma at a total protein concentration of 0.25%. This process strategy also allowed simple operation and easy of affinity ligand recycling.