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

Abstract

Background: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. Objectives: 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. Materials and Methods: 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 QSFF with 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. Results: 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 -1 of resin as the dynamic binding capacity. Conclusions: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).

Investigation of purification process stresses on erythropoietin peptide mapping profile

“…The cell culture process was explained in a previous work. [ 48 ] Fifty milliliters of CHO cell culture harvest was applied, to gel filtration chromatography XK 26/40 column (GE Healthcare, Uppsala, Sweden,+46-18) packed with 175 mL Sephadex G25 media (GE Healthcare, Uppsala, Sweden, +46-18) pre-equilibrated with sodium acetate pH 5 buffer at 3.5 mL/min flow rate by a semi-preparative high performance liquid chromatography (HPLC) system (Waters, Milford, USA, +1-508). The eluted protein was applied to a XK50/20 column (GE Healthcare, Uppsala, Sweden, +46-18) packed with 60 mL anion exchange chromatography Q-Sepharose fast flow media (GE Healthcare, Uppsala, Sweden, +46-18) pre-equilibrated with above-mentioned buffer at 19.6 mL/min flow rate.…”