SYSTEMATIC APPROACH TO CHARACTERIZE AND DEVELOP A CELL FREE GLYCOPROTEIN REMODELING PLATFORM
Glycosylation has profound impacts in many biological processes, such as immune response, inflammation, cell- cell communication, and host- pathogen interaction. The importance of glycosylation is further accentuated as more than 70% of approved protein-based drugs are asparagine-linked (N-linked) glycoproteins. In many cases, the efficacy, safety, and stability of glycoprotein drugs are dictated by its glycan structure. Despite its importance, advancement in glycoscience and glycoengineering is hindered, largely due to insufficient understanding of glycan biosynthesis and bioprocess, as well as, a lack of platform for producing homogeneous designed glycan structures on proteins. Current glycoprotein expression platforms, such as using Chinese hamster ovary (CHO) cell line, cannot avoid heterogeneous glycoform with high batch to batch variability due to their endogenous glycosylation pathways that are sensitive to a change in culture environment. Moreover, the complexity of metabolic network inside cells has made it difficult to understand and engineer specific glycosylation pathways to produce designer glycoproteins. To address those challenges systematically, we proposed to integrate experimental and mathematical methods, such as E. coli- based cell free glycoprotein synthesis (CFGpS) and constrained based flux balance analysis (FBA) to characterize and optimize glycoprotein synthesis. This platform will in the future allow us to robustly and systematically engineer novel glycosylation pathways. To approach that, we first developed a platform for enzymatically remodeling glycan structures on therapeutic relevant N- glycoproteins. This technology allowed us to have direct control of the glycoform and to produce homogeneous glycoproteins for structure- function relationship study. As a whole, we anticipated our mathematical and experimental approach could facilitate the fundamental understanding in glycoscience and could provide a novel approach for producing glycoprotein therapeutics.
Varner, Jeffrey D.
M.S., Chemical Engineering
Master of Science
dissertation or thesis