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Off‐target glycans encountered along the synthetic biology route toward humanized N‐glycans in Pichia pastoris

Abstract : The glycosylation pathways of several eukaryotic protein expression hosts are being engineered to enable the production of therapeutic glycoproteins with humanized application‐customized glycan structures. In several expression hosts, this has been quite successful, but one caveat is that the new N‐glycan structures inadvertently might be substrates for one or more of the multitude of endogenous glycosyltransferases in such heterologous background. This then results in the formation of novel, undesired glycan structures, which often remain insufficiently characterized. When expressing mouse interleukin‐22 in a Pichia pastoris (syn. Komagataella phaffii) GlycoSwitchM5 strain, which had been optimized to produce Man5GlcNAc2N‐glycans, glycan profiling revealed two major species: Man5GlcNAc2 and an unexpected, partially α‐mannosidase‐resistant structure. A detailed structural analysis using exoglycosidase sequencing, mass spectrometry, linkage analysis, and nuclear magnetic resonance revealed that this novel glycan was Man5GlcNAc2 modified with a Glcα‐1,2‐Manβ‐1,2‐Manβ‐1,3‐Glcα‐1,3‐R tetrasaccharide. Expression of a Golgi‐targeted GlcNAc transferase‐I strongly inhibited the formation of this novel modification, resulting in more homogeneous modification with the targeted GlcNAcMan5GlcNAc2 structure. Our findings reinforce accumulating evidence that robustly customizing the N‐glycosylation pathway in P. pastoris to produce particular human‐type structures is still an incompletely solved synthetic biology challenge, which will require further innovation to enable safe glycoprotein pharmaceutical production.
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Soumis le : mercredi 24 mars 2021 - 10:28:20
Dernière modification le : mercredi 23 mars 2022 - 15:51:29

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Bram Laukens, Pieter P. Jacobs, Katelijne Geysens, Jose Martins, Charlot de Wachter, et al.. Off‐target glycans encountered along the synthetic biology route toward humanized N‐glycans in Pichia pastoris. Biotechnology and bioengineering, 2020, Biotechnology and bioengineering, 117 (8), pp.2479-2488. ⟨10.1002/bit.27375⟩. ⟨hal-03179097⟩



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