title: Glyco-engineered HEK 293-F cell lines to produce therapeutic  glycoproteins with human N-glycosylation and improved pharmacokinetics
creator: Uhler, Rico
subject: ddc-570
subject: 570 Life sciences
subject: ddc-600
subject: 600 Technology (Applied sciences)
subject: ddc-610
subject: 610 Medical sciences Medicine
description: Using human cell lines for the production of therapeutic proteins can be beneficial due to their ability to  generate fully human post-translational modifications. HEK 293 cells are an efficient expression system  with a record of approved therapeutic protein products. However, the presence of  N-acetylgalactosamine, the low sialylation on N-glycans of recombinant HEK 293 cell-derived  glycoproteins and the resulting efficient clearance via glycan receptors in patients—namely the  asialoglycoprotein receptor and the mannose receptor—currently limits its use in therapeutic protein  production.  To be able to produce recombinant proteins without N-acetylgalactosamine on their N-glycans,  HEK 293-F clones featuring a functional KO of both the B4GALNT3 and B4GALNT4 genes were  generated. Subsequently, to increase the sialylation, sialyltransferases ST6GAL1 and ST3GAL6 were  overexpressed by gene knock-in. The model protein factor VII-albumin was expressed in these new host  cell lines to evaluate the phenotypic changes induced by the described glyco-engineering. Furthermore,  medium supplementation with N-acetylmannosamine and cytidine was tested to improve sialylation.  HEK 293-F cells with B4GALNT3 and B4GALNT4 knock-out produced factor VII-albumin devoid of  N-acetylgalactosamine and sulfated N-acetylgalactosamine, with reduced antenna fucosylation and  increased antennarity, bisecting N-acetylglucosamine as well as sialylation. Overexpression of  ST6GAL1 or ST3GAL6 further increased sialylation and reduced antenna fucosylation, while  sialylation was not affected by medium supplementation of sialic acid metabolism precursors  N-acetylmannosamine and cytidine. Factor VII-albumin produced in the double N-acetylgalactosamine  transferase knock-out cell line with simultaneous ST6GAL1 knock-in showed a level of sialylation  similar to that of plasma-derived factor VII and higher than that of factor VII from CHO or BHK cells.  As a result, asialoglycoprotein and mannose receptor binding of glyco-engineered factor VII-albumin  variants was abolished in vitro and pharmacokinetic properties were improved in vivo.  To determine whether the detected changes in N-glycosylation were specific for factor VII-albumin or  whether the results are applicable to other proteins, B domain-deleted factor VIII and factor IX were  expressed in the wild-type HEK 293-F cell line, the double N-acetylgalactosamine transferase knockout  clone and the double N-acetylgalactosamine transferase knock-out clone with ST6GAL1 knock-in.  Despite considerable differences in the N-glycosylation profiles of the three proteins, the effects of the  applied glyco-engineering were largely comparable.  Thus, these new glyco-engineered cell lines are suitable for the expression of fully human therapeutic  proteins with favorable N-glycosylation and improved pharmacokinetic properties.
date: 2022
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/31087/1/PhD%20Thesis%20Rico%20Uhler.pdf
identifier: DOI:10.11588/heidok.00031087
identifier: urn:nbn:de:bsz:16-heidok-310877
identifier:   Uhler, Rico  (2022) Glyco-engineered HEK 293-F cell lines to produce therapeutic glycoproteins with human N-glycosylation and improved pharmacokinetics.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/31087/
rights: info:eu-repo/semantics/openAccess
rights: Please see front page of the work (Sorry, Dublin Core plugin does not recognise license id)
language: eng