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Off‐target glycans encountered along the synthetic biology route toward humanized N ‐glycans in Pichia pastoris
Author(s) -
Laukens Bram,
Jacobs Pieter P.,
Geysens Katelijne,
Martins Jose,
De Wachter Charlot,
Ameloot Paul,
Morelle Willy,
Haustraete Jurgen,
Renauld JeanChristophe,
Samyn Bart,
Contreras Roland,
Devos Simon,
Callewaert Nico
Publication year - 2020
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.27375
Subject(s) - pichia pastoris , glycan , exoglycosidase , glycosylation , glycosyltransferase , biochemistry , biology , pichia , glycoprotein , glycomics , n linked glycosylation , heterologous expression , golgi apparatus , computational biology , recombinant dna , gene , endoplasmic reticulum
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 Man 5 GlcNAc 2 N ‐glycans, glycan profiling revealed two major species: Man 5 GlcNAc 2 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 Man 5 GlcNAc 2 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 GlcNAcMan 5 GlcNAc 2 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.