Development of a Methodology for the Quantification of N-Glycosylation Fluxes with Glycosylation Kinetic Flux Profiling (GlyKiF)

N-glycosylation, a critical post-translational modification that can have a significant impact on biologic stability and efficacy, occurs through a complex branched network of fluxes that can be challenging to analyze and quantify. The main aim of this work was to develop a method for measuring the fluxes for a biantennary N-glycosylation network that relies on the use of stable isotope labeled sugars through an application of kinetic flux profiling (KFP), called Glycosylation-KFP (GlyKiF). The development of the methodology involved optimizations for the experimental steps as well as improvements to the computational scripts required for flux prediction. Glycosylation fluxes were estimated for recombinant CHO cells secreting an IgG antibody for two different process conditions. Significant differences in fluxes were identified at the Man5 secretion step and with fluxes related to galactosyltransferase activity. These results serve as a proof of concept for applications of KFP in the glycosylation sphere, expanding the capabilities of stable isotopes in determining fluxes of various previously untargeted metabolic networks. Through GlyKiF, glycosylation networks can be constructed for multiple cell lines and cell line specific differences can be detected, allowing for the identification of bottlenecks in the network and serving as a guide for further glycoengineering endeavors