Experimental Methods Towards Controlling the Glycoform

The glycosylation profiles of biopharmaceuticals have a major impact on the bioactivity, bioavailability, biocompatibility and biosafety of the therapeutic. As modulation of the glycosylation profile can enhance or attenuate therapeutic properties of the drug, methods towards control of glycosylation therefore are of interest to the research, clinical and industrial communities. This work embarks on a multipronged approach towards achieving uniform, consistent and specified protein glycosylation through development of analytical methodologies to monitor attributes of glycosylation (Chapters 2-4), and by construction of a cell-free bioreactor for In-Vitro Glyco-Engineering (Chapter 5). In objective (1) development of state-of-the-art analytical technologies demonstrated how Chinese Hamster Ovary (CHO) cells, subjected to high-flux substrates and cellular and protein engineering strategies, can be manipulated towards achieving control over the protein glycosylation pathway in mammalian cells. HPLC-based methods were employed to determine: amino acid composition, glycosylation patterns, nucleotide sugar distributions and titer. Modulation of the feed was shown to increase nucleotide sugars, specifically CMP-Neu5Ac, resulting in increased sialylation of proteins. This detailed representation facilitated fundamental insight into the physiology of cellular responses, by synergizing how the glycosylation profile is inextricably coupled to substrate availability, enzyme activities and cellular metabolism, thereby revealing the interplay of the glycosylation processes at the systems level. In objective (2) implementation of a novel, transformative and scalable biofabrication process applying In-Vitro Glyco-Engineering of glycoproteins to produce structurally well-defined, homogeneous glycans. This chemo-enzymatic approach utilizes Endoglycosidase-H covalently coupled to a solid phase support of silicon nanowires to deglycosylate proteins. Select model glycoproteins: RNaseB, alpha-amlyase and IgG, showed the flexibility of the process towards different classes of biomolecules. These efforts will lead to higher productivity, increased product homogeneity and greater therapeutic efficacy. Ultimately, to enable the cost effective and routine production of biopharmaceuticals to facilitate increased patient affordability and larger profit margins. Advisor: Michael Betenbaugh Reader: Kevin Yarem