Aims The present study was aimed at design of experiments (DoE)- and artificial intelligence-based culture medium optimization for high level extracellular production of a novel recombinant human interferon alpha 2b (huIFN alpha 2b) in glycoengineered Pichia pastoris and its characterization. Methods and Results The artificial neural network-genetic algorithm model exhibited improved huIFN alpha 2b production and better predictability compared to response surface methodology. The optimized medium exhibited a fivefold increase in huIFN alpha 2b titre compared to the complex medium. A maximum titre of huIFN alpha 2b (436 mg l(-1)) was achieved using the optimized medium in the bioreactor. Real-time capacitance data from dielectric spectroscopy were utilized to model the growth kinetics with unstructured models. Biological characterization by antiproliferative assay proved that the purified recombinant huIFN alpha 2b was biologically active, exhibiting growth inhibition on breast cancer cell line. Conclusions Culture medium optimization resulted in enhanced production of huIFN alpha 2b in glycoengineered P. pastoris at both shake flask and bioreactor level. The purified huIFN alpha 2b was found to be N-glycosylated and biologically active. Significance and Impact of the Study DoE-based medium optimization strategy significantly improved huIFN alpha 2b production. The antiproliferative activity of huIFN alpha 2b substantiates its potential scope for application in cancer therapy.