Subcellular Fractionation Enables Assessment of Nucleotide Sugar Donors Inside the Golgi Apparatus as a Prerequisite for Unraveling Culture Impacts on Glycoforms of Antibodies.

Glycosylation is a critical quality attribute in biopharmaceuticals that influences crucial properties, such as biological activity and blood clearance. Current methods for modeling glycosylation typically rely on imprecise or limited data on nucleotide sugar donor (NSD) dynamics. These methods use in vitro transporter kinetics or flux balance analysis, which overlook the key aspects of metabolic regulation. We devised an integrative workflow for absolute subcellular NSD quantification in both cytoplasm and secretory organelles. Using subcellular fractionation, exhaustive sample extraction, and liquid chromatography triple-quadrupole tandem mass spectrometry, we accurately measured NSD concentrations ranging from 1.6 amol/cell to 3 fmol/cell. As expected, NSD concentration profiles aligned closely with the glycan distributions on antibodies, particularly after nutrient pulsing to stimulate NSD production, showcasing method validity. This method enables empirical observation of compartment-specific NSD dynamics. Thus, this study provides novel insights indicating that N-glycosylation, which governs NSD supply, is primarily regulated within the Golgi apparatus (GA). This method offers a novel tool to obtain sophisticated data for a more efficient optimization of glycosylation processes in production cell lines.