Optimization of Dynamic Binding Capacity of Anion Exchange Chromatography Media for Recombinant Erythropoietin Purification

Abstract

<span style="color: windowtext;">Background:</span><span style="color: windowtext;">The dynamic binding capacity (DBC) of a chromatography matrix in protein purification is the amount of the total protein absorbed into the matrix, before occurrence of a significant break in the breakthrough curve. Optimization of the process criteria for maximum DBC avoids extra process scale-up and reduces the processing time, costs and protein loss. Taguchi method is a simple useful tool in experimental design to estimate the optimal condition with minimum experiments.</span> <span style="color: windowtext;">Objectives:</span><span style="color: windowtext;">In this research, linear flow rate, pH and protein concentration of the feed were checked according to an L9 orthogonal Taguchi array, to estimate the best conditions for maximum DBC of Q-sepharose fast flow (QSFF) resin in recombinant human erythropoietin purification process.</span> <span style="color: windowtext;">Materials and Methods:</span><span style="color: windowtext;">A crud sample containing human recombinant erythropoietin was harvested from a cell culture of Chinese hamster ovary (CHO) cell line. Desalted harvests with different total protein concentrations (30, 40 and 50 µg.mL^-1) and pH values (5, 6 and 7) were loaded into a packed column of QSFFwith different linear flow rates (60, 120 and 280 cm.h^-1) up to 10% of the breakthrough curve. The total protein loading to the column was checked by UV absorbance and Lowry method, and erythropoietin concentration was measured by ELISA. Analysis of variance (ANOVA) was applied to determine the optimum condition.</span> <span style="color: windowtext;">Results:</span><span style="color: windowtext;">Finally, total protein concentration of 50 µg.mL^-1, pH of 5 and flow rate of 120 cm.h^-1, were anticipated as the optimal process conditions with 5.85 mg.mL^-1of resin as the dynamic binding capacity.</span> <span style="color: windowtext;">Conclusions:</span><span style="color: windowtext;">Experiments with anticipated optimal criteria were performed three times and no significant difference was observed (p = 0.136, and 6.06 mg/mL as the average dynamic binding capacity).</span>