Abstract
Monoclonal antibody N-glycosylation is a critical quality attribute influencing therapeutic safety and efficacy, and is strongly influenced by bioprocess design. NISTCHO, a publicly available Chinese hamster ovary producer cell line, is increasingly encouraged for use as a reference system. However, the impact of feeding strategies on cellular performance and N-glycosylation has not been assessed. Here, we applied multivariate analysis of compositional N-glycan data to assess how feeding strategies influence N-glycan composition of cNISTmAb. We varied feeding strategies in frequency, glucose supply, and galactose/manganese supplementation. Feeding frequency had minimal impact on quality attributes but strongly affected culture performance, with every-other-day feeding improving titers and cell-specific productivity. High glucose availability supported growth and productivity. Low glucose strategies reduced titers and shifted N-glycosylation towards non-galactosylated and fucosylated species, despite lactate accumulation remaining within favorable ranges. Galactose and manganese consistently increased antibody galactosylation, with galactose additionally serving as an auxiliary carbon source, extending cell viability. Importantly, mAb glycation remained stable across all feeding strategies at harvest. Overall, these results demonstrate that feed composition and timing can be used to tune both cellular performance and mAb glycosylation, establishing NISTCHO as a robust benchmark for standardized process-quality studies.
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Data availability
All raw data generated during the bioprocess analyses, including raw mass spectrometry files and a detailed fed-batch protocol (for all feeding strategies), have been deposited in Zenodo and are publicly available at https://doi.org/10.5281/zenodo.17046013.
Code availability
Full analysis code for analysis reproducibility is available at https://github.com/csbg/DGTX_feeding_strategies_nistcho.
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Acknowledgements
We would like to thank Julian Rabl for the initial measurements of the test data as part of his practical lab training. The authors want to thank Christof Regl for providing valuable commentary to the manuscript. We would like to acknowledge Anja Horger and Andreas Fellner for scientific guidance of the manganese and galactose measurements. Thanks to Markus Riedl for help in data visualization. The Biolayer interferometer– Octet RED96e (Sartorius) device was kindly provided by the Connective Base GmbH, and the project was supported by the BOKU Core Facility Biomolecular Cellular Analysis. Lastly, the authors would like to thank Alexandar Paunovic and Ursula Kiesswetter for their excellent technical support. This research was funded in whole or in part by the Austrian Science Fund (FWF) 10.55776/FG12 [”DigiTherapeutX”]. For open access purposes, the author has applied a CC BY public copyright license to any author accepted manuscript version arising from this submission. The authors acknowledge the use of OpenAI’s ChatGPT (GPT-5) to assist in language polishing and formatting of the manuscript. All intellectual content, analysis, and conclusions are the authors’ own responsibility.
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Conceptualization: V.S., L.H., T.B., J.Š., N.M., T.R., W.E.S., D.H., L.L., P.F., R.B.G., C.G.H., N.B., and N.F. Methodology: V.S., L.H., T.B., and P.F. Formal analysis: V.S. and L.H. Investigation: L.H. and T.B. Resources: V.S., L.H., and T.B. Data curation: V.S. and L.H. Writing—Original Draft: V.S., L.H., and T.B. Writing—review and editing: V.S., L.H., T.B., J.Š., N.M., T.R., W.E.S., D.H., L.L., P.F., R.B.G., C.G.H., N.B., and N.F. Visualization: V.S. and L.H. Supervision: V.S., R.B.G., C.G.H., N.B., and N.F. Project administration: V.S., R.B.G., C.G.H., N.B., and N.F. Funding acquisition: R.B.G., C.G.H., N.B., and N.F.
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Schäpertöns, V., Hofer, L., Berger, T. et al. Effects of feeding strategies on culture performance and product quality in NISTCHO. npj Syst Biol Appl (2026). https://doi.org/10.1038/s41540-026-00686-3
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DOI: https://doi.org/10.1038/s41540-026-00686-3
