Glycosylation pattern is an important quality attribute of therapeutic proteins. It affects protein stability, half-life and even biological activity. In the production of therapeutic proteins, the glycan pattern must be consistent among the products produced over time, in different sites and even by different manufacturers. Several approaches are being used to modulate glycan profiles to enhance a favorable characteristics or to meet the product specification. In this study, we employed a systematic approach to engineer CHO cells with desirable glycosylation machinery, allowing more robust control of glycosylation patterns.<br/>We employ a mathematical model which incorporates reaction rules and enzyme kinetics of N-glycosylation pathway. The model is used to trace all possible reaction paths leading to the N-glycan profile of protein produced in a recombinant CHO-K1 cell line and then, identify potential gene targets for glycoengineering. These targets include glycosyltransferases, glycosidases or genes involved in the synthetic pathways and Golgi transport of nucleotide sugars. This pathway analysis also reveals possible gene combinations that might enrich particular glycan species in the glycan profiles. The transcriptome data is used for the selection of promoters with proper strength to drive the expression of these genes. Upon the gene transfection into the producing cell line, the glycan profiles are analyzed by HPLC using 2AB-labeling method. The resulting glycan profile is then compared to the model prediction to refine our strategies of glycoengineering. This systems approach in glycoengineering, integrating transcriptome analysis, model prediction and genetic intervention, facilitates the identification of genetic perturbations necessary to modulate the N-glycosylation pattern of a recombinant protein.

Systems engineering of N-glycans for the production of recombinant proteins in mammalian cells

253rd National Meeting (2017)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.