A novel low-parameter computational model to aid in-silico glycoengineering
© Spahn et al. 2015
Published: 14 December 2015
We use the COBRA toolbox to generate an in-silico representation of the N-glycosylation network. The stochastic transition of glycans through this reaction network is modeled as a Markov chain where secreted glycans are represented as absorbing states (Figure 1B,C). After the user has submitted an experimentally derived glycoprofile on a specific protein (for instance, obtained from a cell culture grown under standard conditions, Figure 1C), sampling methods are used to deduce the unknown probabilities of transitioning from one glycan to another in the network. These transition probabilities are concisely assembled in a Markov transition matrix (Figure. 1D). After this fitting procedure, enzyme knockouts are modelled by setting particular transition probabilities to zero and adjusting the remaining probabilities through optimization (Figure 1E).
Our model is capable of creating N-glycosylation reaction networks that are complex enough to cover typical glycoprofiles found in biopharmaceutical manufacturing including tetra-antennary, highly sialylated or polylactosamine carrying glycans. The probabilistic framework implemented in this model proves to outperform knockout predictions derived from pure constraints-based modeling. Tests on experimental knockout glycoprofiles both from the literature and our laboratory show that the model yields sound predictions of glycoprofile change upon genetic modification which are in good congruence with corresponding experiments (Figure 1F).
Conclusion and outlook
The model has the potential to provide a cheap and fast guidance tool to help find host conditions that can yield a desired glycoprofile, thus providing an important step towards the in-silico process of glycoengineering. So far, the reaction network considered is specific for CHO cells but can be easily modified to include reactions occurring in other hosts. In addition, it could be integrated into whole-cell metabolic models. This would enable comprehensive in-silico representations of the entire cell-culture setup, allowing one to simulate the effects on the glycoprofile of a wide range of both intracellular and extracellular modifications to the growth conditions.
This work was funded from a generous gift from the Novo Nordisk Foundation to the Center for Biosustainability. A provisional patent has been filed concerning this work. In addition, we wish to thank H. Clausen and his group from Copenhagen University for valuable discussions and sharing unpublished data.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.