Process development for the production of recombinant human interferon gamma (hifn-Γ) in pichia pastoris cell factory
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Human interferon gamma (hIFN-γ) is a pleiotropic cytokine that is produced by natural killer cells and T lymphocytes. hIFN-γ plays an key role in communicating innate and acquired immune systems during bacterial and viral infections, it also possess antiviral, immunoregulatory, and anti-tumor properties. In the present study, cell level and process level strategies were applied to address the expression machinery related and process related problems, which are bottle neck for protein expression in Pichia pastoris. Co-expression of chaperones and codon optimization enhanced the production of hIFN-γ to 2.5 mg/L. Further medium development for high level expression of hIFN- γ from Pichia pastoris (GS115) was performed with the aid of statistical and nonlinear modeling techniques. Sequential optimization of modified FM22 medium with RSM and ANN-GA resulted in 30 mg/L of hIFN-γ production. The validation was carried out in batch bioreactor and unstructured kinetic models were adapted. The Luedeking-Piret (L-P) model showed production of hIFN-γ was mixed growth associated with the maximum production rate of 40 mg/L of hIFN-γ production. . Different substrate inhibition models were fitted to the growth kinetic data and the additive form of double webb model was found to be the best to explain the growth kinetics of recombinant P.pastoris. A novel purification strategy for recombinant human interferon gamma (rhIFN-γ) using nickel chelated metal affinity reverse micellar extraction was demonstrated. The development of this purification system with optimized parameters led to an efficient recovery of 67.3% and improved purity of 79.54%. The anti-proliferative activity on A431 cell lines showed that 50 % inhibition with 80 ng/ml rhIFN-γ concentration and the cells showed necrotic activity.
Supervisor: Venkatadasu Veeranki
BIOSCIENCES AND BIOENGINEERING