(507a) Glycosylation-on-a-Chip for Tunable Cell-Free Synthesis of Glycoproteins

A key feature in the development of new therapeutics is glycosylation, a post-translational modification where a complex sugar group, called a glycan, is attached to a protein. Glycans have diverse chemical structures that affect drug properties such as targeting, biological activity, and immunogenicity. Currently, the standard platform to produce glycoprotein therapeutics are Chinese hamster ovary (CHO) cells because they have native cellular machinery required for glycosylation. However, this native machinery is prone to making heterogeneous glycoprotein structures where it can be difficult to isolate specific glycoprotein structures from the array of intermediate glycoforms and side products. Furthermore, glycosylation is an important part of cellular function, so any changes to existing glycosylation machinery can limit or even inhibit cell growth. To address these challenges, cell-free technologies have emerged that aim to decouple protein synthesis and post-translational modifications from cell growth.

In this talk, I will describe a microfluidic platform that seamlessly integrates cell-free protein synthesis (CFPS), glycosylation, and purification of a model glycoprotein. Microfluidics offer advantages such as reaction compartmentalization, tunable residence time, reusability of tethered enzymes, and the potential for continuous manufacturing. Moreover, it affords an unprecedented opportunity for spatiotemporal control of glycosylation reactions that is difficult or impossible to achieve with existing cell-based and cell-free glycosylation systems. As proof-of-concept, super-folder green fluorescent protein (sfGFP) was used as substrate, enabling both the protein production and purification processes to be visualized and easily quantified during optimization of the microfluidic system. In the first compartment of the device, the sfGFP protein was expressed by Escherichia coli cell-free extract, which efficiently catalyzed transcription and translation of the target protein on chip. Next, the newly expressed sfGFP, which carried a C-terminal glycosylation tag for glycan attachment, was delivered to a second on-chip compartment where it was subjected to glycosylation machinery. Glycosylation of sfGFP was achieved using components from a well-characterized bacterial asparagine-linked (N-linked) glycosylation pathway derived from Campylobacter jejuni that involves the oligosaccharyltransferase PglB as the conjugating enzyme and lipid-linked heptasaccharide glycan as the donor substrate. Specifically, PglB, an integral membrane protein, was biotinylated and immobilized in the device using biotin and streptavidin interactions, thereby enabling reuse of this important biocatalyst. Lastly, in a third compartment, the sfGFP product was isolated using a nickel-coated surface that facilitated affinity-capture of the hexahistidine-tagged protein. Overall, this work describes a first-in-kind “glycosylation-on-a-chip” prototype that could find use as a laboratory tool for mechanistic dissection of the protein glycosylation process as well as a biomanufacturing platform for small-batch, decentralized glycoprotein production.