Feeding strategies in recombinant human erythropoietin production by Pichia pastoris fermentation in stirred-tank bioreactor
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Date
2014-08
Authors
Mohd Ridhwan, Shahkillahwati
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Abstract
Administration of erythropoietin or EPO as a therapeutic improves the quality of life in patients with cancer, renal and anemia. Knowing the importance of EPO and the high demand in the biopharmaceutical market, Pichia pastoris strains GS115 2 (5) has been genetically engineered to produce recombinant human erythropoietin (rhuEPO) from the synthetic gene using three-stage cultivation process. The culture was first grown in production medium containing glycerol as a carbon sources (phase I) prior to obtained high cell density culture (phase II) and was induced with methanol for protein production (phase III). Medium formulation and enhanced batch cultivation was investigated prior to fed-batch fermentation. Various feeding strategies and methanol induction conditions which highly influenced the production of protein were also investigated. A process of high cell density cultivation was developed which took 60 hours for the culture to reach seven fold of cells 132.10 g/L using constant flow rate. An improved induction condition was also developed using step wise increase feeding strategy. It took only 19 hours from the induction phase to produce a total of 104.8 ng/L of EPO with the yield of product over biomass (Y p/x) 1.19 ng g-1 compared to the preliminary 36 hours.
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Stirred-Tank Bioreactor