High-throughput profiling of nucleotides and nucleotide sugars to evaluate their impact on antibody N-glycosylation

• Modulation of mAb N-linked glycosylation in micro-bioreactors using different feed additives.
• A high-throughput MALDI-TOF-MS technique reduced the analysis time for intracellular nucleotides and nucleotide sugars more than one order of magnitude.
• UDP-Gal synthesis appeared to be limiting, however a further increase of UDP-GlcNAc or GDP-Fuc left the glycosylation process mostly unchanged.
• Manganese and asparagine supplementation significantly altered the glycosylation profiles without changing intracellular components.
• Scalability was demonstrated by a pilot-scale bioreactor.

Recent advances in miniaturized cell culture systems have facilitated the screening of media additives on productivity and protein quality attributes of mammalian cell cultures. However, intracellular components are not routinely measured due to the limited throughput of available analytical techniques. In this work, time profiling of intracellular nucleotides and nucleotide sugars of CHO-S cell fed-batch processes in a micro-scale bioreactor system was carried out using a recently developed high-throughput method based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF-MS). Supplementation of various media additives significantly altered the intracellular nucleotides and nucleotide sugars that are inextricably linked to the process of glycosylation. The results revealed that UDP-Gal synthesis appeared to be particularly limiting whereas the impact of elevated UDP-GlcNAc and GDP-Fuc levels on the final glycosylation patterns was only marginally important. In contrast, manganese and asparagine supplementation altered the glycan profiles without affecting intracellular components. The combination of miniaturized cell cultures and high-throughput analytical techniques serves therefore as a useful tool for future quality driven media optimization studies.