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

## TL;DR

This study introduces a new method to measure nucleotide sugar donor levels in the Golgi apparatus, improving understanding of how cell culture affects antibody glycosylation.

## Contribution

A novel workflow for subcellular quantification of nucleotide sugar donors in the Golgi apparatus and cytoplasm.

## Key findings

- NSD concentration profiles matched glycan distributions on antibodies after nutrient pulsing.
- N-glycosylation is primarily regulated within the Golgi apparatus.
- The method enables detailed observation of NSD transport mechanisms in Chinese hamster ovary cells.

## Abstract

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.

A novelty analytical workflow for quantitative investigation of transport mechanisms governing the nucleotide sugar supply from the cytoplasm to the Golgi apparatus for N‐glycosylation of antibodies in Chinese hamster ovary cells was developed. The workflow enabled compartment‐specific quantification of nucleotide sugar concentrations within the cytoplasm and the secretory pathways which aligned with nutrition‐induced changes in glycosylation patterns on produced antibodies, demonstrating the efficacy of the method. With this method, we were able to illuminate the transport mechanisms of NSDs within the cell, indicating that the supply of nucleotide sugars into the Golgi apparatus is to a large extent regulated by metabolite concentrations within the Golgi.