Metabolic engineering of insect & mammalian cells to improve product quality.

摘要

An increasing percentage of drugs today are biologically derived and the cells used to produce these molecules are the micro-reactors of today's pharmaceutical industry. Hence, it is critical to optimize these cells and engineer the metabolic pathways within for superior product quality and yields.; Many of these drugs are glycoproteins and the N-glycan structures on them strongly influence their bioactivity and in vivo half life. Despite the high product yields possible using baculovirus-insect cell expression system, insect cells are not yet ideal for glycoprotein production due to undesirable N-glycan structures. The beta-N-acetylglucosaminidase activity in insect cells presents a major hindrance towards efficient generation of homogeneous mammalian-like glycan structures. In a study presented in this thesis (Ch. 2), we have reported successful identification, purification and characterization of a novel beta-N-acetylglucosaminidase (SfHex) of insect origin that is capable of removing beta(1,2)- N-acetylglucosamine from an N-glycan. Yet another reason limiting the use of insect cells for producing mammalian-like glycoproteins is their inability to synthesize sialic acid. The study here (Ch. 3) reports a strategy to generate enhanced levels of CMP-sialic acid in insect cells through expression of a mutant UDP-GlcNAc-2-epimerase/ManNAc kinase.; The glycan structures and their heterogeneity vary with species. This may be due to difference in levels of enzymes capable of modifying glycan structures. Knowledge of these levels would enable a wiser choice of cell lines and towards this, a study comparing the levels of exoglycosidases in insect and mammalian cell lines is presented in Chapter 4. Variation in glycan structures with species has important bearing in choosing cell lines to produce diagnostic proteins. The study presented here (Ch. 5) revealed that diagnostic glycoproteins produced in High Five(TM) cells are likely to give false positives and decrease assay specificity for parasitic diseases.; Finally, mammalian cells incorporating glutamate synthetase (GS) selection system are commonly used in today's industry. However, the GS-system may disturb various metabolic pathways causing poor product quality and quantity. In Chapter 6, we have used metabolic engineering to modify intracellular metabolite utilization system and reduce undesired by-products as a result of pyruvylation and oxidation and increase product yields.