| Many important biotherapeutics, such as growth factors, hormones, and antibodies are produced in mammalian cell expression systems because these cells can perform certain post-translational modifications that are vital to the biological and pharmacokinetic properties of the protein product. N-glycosylation is a post-translational modification that involves the production of a lipid-linked oligosaccharide (LLO) and the subsequent transfer of the oligosaccharide onto the polypeptide chain by the oligosaccharide transferase (OST) enzyme complex. In some cases, commercial overexpression of glycoproteins can lead to under-glycosylated products in which not all potential N-glycan sites are occupied. This variable site occupancy can affect the safety and efficacy of the biotherapeutic. Therefore, there is significant interest in furthering our understanding of N-glycan variable site occupancy. Deficiencies in N-glycosylation are also associated with a class of diseases known as Congenital Disorders of Glycosylation (CDG).;In this study, we investigated the capacity of analytical methods to characterize the LLO synthesis pathway, then utilized metabolic engineering strategies in an attempt to alter LLO synthesis. First, a human embryonic kidney cell line expressing human transferrin was evaluated as a potential model system for site-occupancy deficiency. By analyzing the levels of intermediates in the LLO synthesis pathway in Chinese Hamster Ovary (CHO) cells, we determined that multiple factors, such as cell type, media composition, and cell adherence can affect the availability of the LLO donor. CHO cells with mutations affecting the LLO synthesis pathway, MI8-5 and B4-2-1, could potentially be used to develop treatments for CDG. LLO levels in the CHO MI5-4 mutant indicate that low hexokinase activity limits LLO synthesis and that overexpression of hexokinase can improve the availability of the final LLO donor. Finally, enzymes responsible for potential rate limiting steps in the LLO synthesis pathway were overexpressed in CHO cells. Overexpression of STT3A, the catalytic subunit of the OST, had no affect on the availability of the completed LLO donor. Overexpression of the mannosyltransferase ALG3 could potentially increase the mannosylation of LLO intermediates. Increased expression of Rft-1, a protein which may be involved in LLO transport, caused a slight increase in the relative quantity of the final LLO donor. |
