Author/Authors :
-، - نويسنده Recombinant Biopharmaceutical Production Department, Research and Production Complex, Pasteur Institute of Iran, Karaj, I.R. IRAN Sepahi, Mina , -، - نويسنده Recombinant Biopharmaceutical Production Department, Research and Production Complex, Pasteur Institute of Iran, Karaj, I.R. IRAN Kaghazian, Hooman , -، - نويسنده Recombinant Biopharmaceutical Production Department, Research and Production Complex, Pasteur Institute of Iran, Karaj, I.R. IRAN Payravi Sereshkeh, Mina , -، - نويسنده Quality Control Department, Research and Production Complex, Pasteur Institute of Iran, Karaj, I.R. IRAN Sadeghcheh, Tahereh , -، - نويسنده Quality Control Department, Research and Production Complex, Pasteur Institute of Iran, Karaj, I.R. IRAN Hadadian, Shahin , -، - نويسنده Recombinant Biopharmaceutical Production Department, Research and Production Complex, Pasteur Institute of Iran, Karaj, I.R. IRAN Jebeli, Mohammad Reza , -، - نويسنده Science and Research Branch, Islamic Azad University, Tehran, I.R. IRAN Yavari, Fereshteh
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 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.
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-1of 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).
