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

Abstract

Background:The glycosylation pathways of several eukaryotic protein expression hosts are being engineered to enable the production of therapeutic glycoproteins with humanized applicationcustomized 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.Results: When expressing mouse interleukin-22 (mIL-22) in a Pichia pastoris (syn. Komagataella phaffi) GlycoSwitchM5 strain which had been optimized to produce Man5GlcNAc2 N-glycans, glycan profiling revealed two major species: Man5GlcNAc2 and an unexpected, partially αmannosidase-resistant structure. A detailed structural analysis using exoglycosidase sequencing, mass spectrometry, linkage analysis and NMR, revealed that this novel glycan was Man5GlcNAc2 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 GlcNAcMan5GlcNAc2 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 glycoengineered expression host cells should always be accompanied by detailed glycan analysis of the particular therapeutic proteins being produced. Conclusions:Our findings reinforce accumulating evidence that robustly customizing the Nglycosylation pathway in Pichia 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.

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

“…These additional N-glycans (with a main peak of Hex8GlcNAc2) were recalcitrant to α-1,2-mannosidase and were only partially hydrolyzed by Jack Bean α-1,2/3/6-mannosidase. This behavior is identical to that of glycans observed earlier on other P. pastoris 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).…”

“…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).…”

Production of Recombinant Human Ceruloplasmin: Improvements and Perspectives

“…Although SM5 is engineered to produce HexNAc2Hex5 structures, the two most abundant species were high-mannose HexNAc2Hex8 and HexNAc2Hex11. Off-target unexpected N-glycan structures have been detected in other proteins expressed in SM5, indicating that this is probably a common problem encountered with glycoengineered P. pastoris strains [ 37 ]. The dominating structures of these N-glycans were variable and appeared to be somewhat protein-dependent [ 37 ].…”

“…Off-target unexpected N-glycan structures have been detected in other proteins expressed in SM5, indicating that this is probably a common problem encountered with glycoengineered P. pastoris strains [37]. The dominating structures of these N-glycans were variable and appeared to be somewhat protein-dependent [37]. Determination of glycosite occupancy was performed by analysis of tryptic glycopeptides after their enrichment by HILIC SPE (Figure 4).…”

Industrial Production of Proteins with Pichia pastoris—Komagataella phaffii

“…This species is widely applied as a heterologous protein production host, and its utilization has been widely reported in the literature [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 ]. The main advantages of this organism are the possibility to run high-density fermentation according to established protocols, fast-paced and automation-friendly genetic engineering [ 10 ], eukaryotic post-translational modifications [ 11 , 12 ], high secretory efficiency and biomass yields [ 13 , 14 ], stable genetic constructs [ 15 ], and an increasing collection of publicly available tools [ 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 ].…”