Development of a bioprocess for recombinant protein production with a Pichia pastoris och1 knock-out strain

Abstract

The methylotrophic yeast Pichia pastoris is a widely used host organism for recombinant protein production in biotechnology and biopharmaceutical industry. However, if the target product describes a glyco-protein, the yeast has the tendency for hypermannosylation triggered by the enzyme a-1,6-mannosyltransferase. The resulting heterogeneous and vast glycosylation pattern on the surface of the recombinant protein hampers traditional downstream processes and limits biopharmaceutical applications. In this Thesis, a controllable bioprocess with a P. pastoris strain, where the responsible gene (och1) for hyperglycosylation was knocked out, was developed. Due to a drastically altered morphology of the P. pastoris och1 knock-out strain compared to the wildtype, various unusual effects were observed: cell lysis, foam formation, a risk of overfeeding and low productivity of the medically relevant recombinant enzyme horseradish peroxidase (HRP). In a multivariate Design of Experiments (DoE) approach, three potential productivity-influencing process parameters - temperature, pH and dissolved oxygen concentration - were analyzed to determine a parameter area for high recombinant protein production. Expression, purification and biochemical characterization of the model protein HRP were also part of this Thesis. Finally, the less-glycosylated enzyme variant, which can be also used for targeted cancer treatment, was analyzed for its reactivity with the prodrug indole-2-acetic acid. A controllable bioprocess for this P. pastoris och1 k.o. strain was successfully developed and with the aid of it an expression of recombinant proteins in a less-glycosylated form is possible. Furthermore, a potential candidate for targeted cancer treatment studies could be determined by analyzing various HRP samples.