(666a) Towards the Cell-Free Expression of Glycoproteins

N-linked glycosylation, the covalent linkage of an oligosaccharide to an amido-group of an asparagine in a consensus sequence motif, is a critical quality attribute in therapeutic proteins that undergo this post translational modification. The biosynthesis and transfer of a carbohydrate chain onto a nascent protein occurs within the ER and these initial steps are conserved throughout eukaryotes. Further processing of the carbohydrate chain occurs in the Golgi apparatus, where differences in processing between species and cell-types arise, resulting in different saccharide composition of the end-product.

These differences are important in nature as N-linked glycosylation has influence over protein function and interaction, but can be problematic when manufacturing a biopharmaceutical. Due to the influence glycan structures have on the pharmacokinetic behaviour of the protein, therefore it is important to ensure a suitable N-linked glycan is produced. This is achieved through manufacturing therapeutic proteins in a suitable expression host (such as CHO) and tightly regulating environmental factors which influence saccharide composition. Nevertheless even with strict parameters in place heterogenous N-linked glycan chains are invariably produced, which can affect the efficacy of a therapeutic protein.

Removing or at least partially removing N-linked glycosylation processing from an in vivo environment, could allow for a enhance user control over the end product. Therefore we have developed a cell-free expression system for the synthesis of glycoproteins. Using a Pichia pastoris cell lysate including microsomes and additional relevant metabolites, we demonstrated the synthesis of a protein with an N-linked oligosaccharide. To investigate the efficiency of site occupancy within a cell-free expression system, we have used a reporter GFP construct containing one N-glycosylation site. This has allowed us to study factors which influence N-linked glycosylation to improve the in vitro system.  Furthermore, we have used a glycoengineering approach to create a ‘humanized’ Pichia pastoris which is capable of producing a more suitable N-linked glycan profile. Our results indicate the successful in vitro synthesis of a glycoprotein with a GlcNAc2Man5 glycan. The GlcNAc2Man5 core structure can be further edited by additional glycosidases and glycosyltransferases. To complement the in vitro core glycosylation, we have designed an in vitro synthetic Golgi reactor to allow further processing of N-linked oligosaccarides towards any user defined structure.

The removal of the N-linked glycosylation process from the constraints of a cell will allow for greater control of the end saccharide composition of the glycan chain. A cell-free protein expression system will reduce heterogeneity allowing for the production of biopharmaceuticals with a homogenesis glycan chain.