Statistical optimization of immunoaffinity purification of hepatitis B surface antigen using response surface methodology

Response surface methodology (RSM) was successfully applied to find an elution condition enhancing the performance of immunoaffinity (IAF) purification of hepatitis B surface antigen (HBsAg), conducted in batch chromatography. The immunosorbent efficiency measured as HBsAg eluted per milliliter of immunosorbent was selected as the response. Two numerical independent variables were studied: concentration of potassium thiocyanate (KSCN) and ethanol, and a categorical variable formed by two levels of sodium deoxycholate under defined ionic strength (DCS/NaCl). A three-level factorial design of RSM was developed and gets a reduced cubic model which accurately fits the data (R2 ≈ 0.97). RSM predicted an optimal region where the response could allow maximum values. At the optimal condition 1.7 M KSCN, 30% (v/v) ethanol, and 0% (w/v) DCS and 1 M NaCl a response of 582 ± 11 μg/ml (559 ± 52 by the model), wherein the measured response does not differs statistically with the maximum experimental value. At optimized condition, a recovery of HBsAg of ∼94% of the adsorbed antigen was obtained, representing a ∼69% more HBsAg than the previously reported. Due to less accumulation of HBsAg this optimum could increase the immunosorbent life in a multiple cycle's operation.