Off-target glycans encountered along the synthetic biology route towards humanized N-glycans inPichia pastoris

Abstract

<jats:title>ABSTRACT</jats:title><jats:sec><jats:title>Background</jats:title><jats:p>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.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>When expressing mouse interleukin-22 (mIL-22) in a <jats:italic>Pichia pastoris</jats:italic> (syn. <jats:italic>Komagataella phaffi</jats:italic>) GlycoSwitchM5 strain which had been optimized to produce Man<jats:sub>5</jats:sub>GlcNAc<jats:sub>2</jats:sub> N-glycans, glycan profiling revealed two major species: Man<jats:sub>5</jats:sub>GlcNAc<jats:sub>2</jats:sub> and an unexpected, partially α-mannosidase-resistant structure. A detailed structural analysis using exoglycosidase sequencing, mass spectrometry, linkage analysis and NMR, revealed that this novel glycan was Man<jats:sub>5</jats:sub>GlcNAc<jats:sub>2</jats:sub> modified with a Glcα-1,2-Manβ-1,2-Manβ-1,3-Glcα-1,3-R tetra-saccharide. Also the biosynthetic intermediates of this off-target modification were detected. 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<jats:sub>5</jats:sub>GlcNAc<jats:sub>2</jats:sub> structure. We have also observed the off-target glycan on other glycoproteins produced in the GlycoSwitchM5 strain. This illustrates the intricacies of Golgi glycosylation pathways and cautions that the use of glyco-engineered expression host cells should always be accompanied by detailed glycan analysis of the particular therapeutic proteins being produced.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>Our findings reinforce accumulating evidence that robustly customizing the N-glycosylation pathway in <jats:italic>Pichia pastoris</jats:italic> 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.</jats:p></jats:sec>

Off‐target glycans encountered along the synthetic biology route toward humanizedN‐glycans inPichia pastoris

“…The reported structure deviates considerably from what was published before (9). Pichia βmannosyltransferases (BMT) are believed to add two consecutive β-1,2-linked mannose residues (6), whereas here a β-1,2-and a β-1,3-linked mannose was present, challenging this hypothesis.…”

“…The N-glycans on glycoproteins produced by P. pastoris typically consist of α-linked mannose residues (21). Previously, an α-mannosidase recalcitrant Hex9GlcNAc2 N-glycan on a recombinant immunoglobulin G (IgG) from glyco-engineered Pichia was described by Gomathinayagam et al (9). Recalcitrance to exoglycosidase digestion was reportedly due to the presence of consecutive β-linked mannose residues, which results from β-1,2-mannosyltransferase-activity that normally occurs on the outer chain of N-glycans (and O-glycans) in wild-type P. pastoris (7,8).…”

“…In addition to exoglycosidase digestion and chemical linkage analysis, 1D and 2D NMRapproaches were used to elucidate more structural details of this N-glycan (6,8,9,23). Although our NMR analysis did not provide full closure about what residue linked the Man5GlcNAc2 with the tetra-saccharide substitution, the combined results of all the analysis allowed us to assign a linear branch-structure consisting of Glcα-1,2-Manβ-1,2-Manβ-1,3-Glcα1-Manα1, with the latter mannose residue attached to the 3-position of one of the terminal α-mannoses of the Man5GlcNAc2 N-glycan.…”

“…Reportedly, such structure has also been found on a human monoclonal IgG produced in glycoengineered P. pastoris (9). The problem could be largely circumvented by identifying and then knocking-out the likely glycosyltransferase culprits.…”

GlycoVHH: Introducing N-glycans on the camelid VHH antibody scaffold - Optimal sites and use for macrophage delivery

“…GlycoSwitchM5-produced proteins, where we identified the Hex8GlcNAc2 species as Man5GlcNAc2 with a substituent consisting of Manβ-1,2-Manβ-1,3-Glcα-1,3-R (Laukens et al, 2020). It is known that the mannosyltransferase gene family is responsible for the addition of -mannose residues in P. pastoris (Mille et al, 2008).…”