PARAMETERS AFFECTING THE EFFICIENCY OF AFFINITY-BASED REVERSE MICELLAR EXTRACTION AND SEPARATION (ARMES) IN GLYCOPROTEIN PURIFICATION

Affinity-based reversed micellar extraction and separation (ARMES) is an effective method for purifying both low and high molecular weight g lycoproteins via liquid-liquid extraction. A range of extraction condi tions were examined to gain insight into the mechanism of ARMES. Conca navalin A (Con A) was used as the model affinity ligand to bind soybea n peroxidase (SEP) and beta-galactosidase as model glycoproteins. Fact orial design was used to investigate the effect of various system vari ables on the extraction of SEP via ARMES. A quadratic model described the system well, resulting in a standard deviation of 7% between calcu lated and experimental extraction efficiencies. Sensitivity analysis s uggested that the key criteria in ARMES were the NaCl concentration an d pH of the aqueous feed phase. Extraction of both glycoproteins decre ased above pH 7 but fell to zero only at pH values significantly above the pi of the model glycoproteins and the Con A affinity ligand. It i s proposed that the complex of the affinity lectin with the glycoprote in results in a sufficiently hydrophobic species that can be extracted into a reversed micellar organic phase even at pH's far above the pI' s of the individual proteins that comprise the complex. This finding h as practical considerations for the use of ARMES in the resolution and purification of protein glycoforms.