PROJECT SUMMARY The glycosylation of therapeutic proteins, such as antibodies, is well known to affect bioactivity, however, uniform glycosylation is challenging to achieve when glycoproteins are produced in bioreactors. One approach to obtaining well-defined homogeneous glycosylation is to use glycosidases and glycosyl transferases (glycoenzymes) to modify the glycosylation of the protein, post-production. This approach is often referred to as in vitro glycoengineering. However, unlike traditional small-molecule chemical synthesis, there are no convenient cost-effective methods or kits for monitoring the progress of the enzyme reactions during glycoengineering. Here, we propose to develop an innovative technology called GlycoSense that will permit in vitro glycoenzyme reactions to be monitored in near real-time using flow cytometry with bead-based glycan- specific reagents. Flow cytometry was chosen as the preferred platform because the data are statistically robust, the equipment is relatively commonplace, the analyses consume very low quantities of sample, and it will enable G&D to leverage its proprietary experience in producing carbohydrate-sensing reagents. In this Phase I application, we will demonstrate the ability of our GlycoSense technology to monitor the effect of glycoenzymes applied to the glycoengineering of commercial therapeutic biologics, provided by a major biopharmaceutical company. These biologics were chosen for the present proof of concept studies because of their medical importance and because of the emerging potential of glycoengineering in the development of biosimilars and biobetters.