Mol, Vivienne (2018) Master thesis: "Development of half-life extended GDF15 for therapeutic applications; expression and secretion in mammalian cells.". Master thesis to be submitted at University Wageningen, Netherlands.

Abstract

With the increasing prevalence of obesity and its related co-morbidities, the necessity for an alternative to surgical intervention is apparent to alleviate the socio-economic burden of the disease on society1. Mediation of satiety and improvement of metabolic conditions in various model species has been shown to be achieved upon treatment with Growth Differentiation Factor (GDF) 15, a potent Transforming Growth Factor-β (TGF-β) superfamily cytokine2–4. To facilitate use of GDF15 as a therapeutic, its short half-life must be extended, and efficient production in mammalian culture supernatant is required. In this research, various protein engineering approaches are applied to obtain the production of secreting, half-life extended GDF15 in mammalian HEK293-FreeStyle culture. Decreasing the promoter strength of various GDF15 constructs did not result in an increased secretion of soluble GDF15. The fusion of a possible chaperone to GDF15 did not result in the detection of secreted, mature GDF15. On the other hand, fusion of a modified Fc domain to GDF15 was shown to significantly increase GDF15 secretion, whilst maintaining GFRAL receptor binding properties of the GDF15. Finally, rational design was used to decrease the cysteine density of the protein in an attempt to favor proper protein folding and hence secretion. Overall, throughout this research more than sixty variant GDF15 constructs were generated and analyzed, yet only one resulted in the significant secretion of half-life extended, mature GDF15. This highlights the difficulty and unpredictability of working with therapeutic proteins in protein engineering purposes.

Item Type:	Article
Date Deposited:	16 May 2018 00:45
Last Modified:	16 May 2018 00:45
URI:	https://oak.novartis.com/id/eprint/35738