Abstract
Recombinant human erythropoietin (rHuEPO) is a critical biopharmaceutical widely used in treating anemia associated with chronic kidney disease and chemotherapy. Current production methods often face challenges related to yield, cost-effectiveness, and consistency. This study focused on optimizing continuous bioprocess parameters for high-titer rHuEPO production using the methylotrophic yeast Pichia pastoris in perfusion bioreactors. A systematic approach investigated the impact of dilution rate, methanol induction strategy, pH, and temperature on cell viability, specific productivity, and rHuEPO titer. Our findings demonstrate that a dilution rate of 0.1 h-1, a controlled methanol feed maintaining a residual concentration below 0.5 g/L, a pH of 5.5, and a temperature of 29°C synergistically led to a significant increase in rHuEPO production. Under these optimized conditions, a sustained rHuEPO titer of 1.2 g/L was achieved over 300 hours, representing a 2.5-fold increase compared to initial baseline conditions, with high cell viability exceeding 95%. This continuous perfusion bioprocess offers a robust and efficient platform for cost-effective, high-yield rHuEPO manufacturing, addressing key limitations of traditional batch and fed-batch systems and contributing significantly to biotechnological innovation in therapeutic protein production.
