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

Paper ID Volume ID Publish Year Pages File Format Full-Text
22698 43370 2016 10 PDF Available
Title
High-throughput profiling of nucleotides and nucleotide sugars to evaluate their impact on antibody N-glycosylation
Abstract

•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.

Keywords
9-AA, 9-aminoacridine; ADP, adenosine diphosphate; AMP, adenosine monophosphate; ATP, adenosine triphosphate; CDP, cytidine diphosphate; CDP, cytidine diphosphate; CMP, cytidine monophosphate; CMP-Neu5Ac, cytidine diphosphate N-acetylneuraminic acid; CTP,
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High-throughput profiling of nucleotides and nucleotide sugars to evaluate their impact on antibody N-glycosylation
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 229, 10 July 2016, Pages 3–12
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us