title: Improvement of N-glycan site occupancy of therapeutic glycoproteins produced in Pichia pastoris.
abstract: Yeast is capable of performing posttranslational modifications, such as N- or O-glycosylation. It has been demonstrated that N-glycans play critical biological roles in therapeutic glycoproteins by modulating pharmacokinetics and pharmacodynamics. However, N-glycan sites on recombinant glycoproteins produced in yeast can be underglycosylated, and hence, not completely occupied. Genomic homology analysis indicates that the Pichia pastoris oligosaccharyltransferase (OST) complex consists of multiple subunits, including OST1, OST2, OST3, OST4, OST5, OST6, STT3, SWP1, and WBP1. Monoclonal antibodies produced in P. pastoris show that N-glycan site occupancy ranges from 75-85 % and is affected mainly by the OST function, and in part, by process conditions. In this study, we demonstrate that N-glycan site occupancy of antibodies can be improved to greater than 99 %, comparable to that of antibodies produced in mammalian cells (CHO), by overexpressing Leishmania major STT3D (LmSTT3D) under the control of an inducible alcohol oxidase 1 (AOX1) promoter. N-glycan site occupancy of non-antibody glycoproteins such as recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) was also significantly improved, suggesting that LmSTT3D has broad substrate specificity. These results suggest that the glycosylation status of recombinant proteins can be improved by heterologous STT3 expression, which will allow for the customization of therapeutic protein profiles.
authors.affiliation: GlycoFi, Biologics Discovery, Merck & Co., Inc, 21 Lafayette St., Suite 200, Lebanon, NH 03766, USA.
authors.forename: Byung-Kwon
authors.initials: BK
authors.lastname: Choi
authors.forename: Shannon
authors.initials: S
authors.lastname: Warburton
authors.forename: Heping
authors.initials: H
authors.lastname: Lin
authors.forename: Rohan
authors.initials: R
authors.lastname: Patel
authors.forename: Istvan
authors.initials: I
authors.lastname: Boldogh
authors.forename: Michael
authors.initials: M
authors.lastname: Meehl
authors.forename: Meehl
authors.initials: M
authors.lastname: Meehl
authors.forename: Marc
authors.initials: M
authors.lastname: d'Anjou
authors.forename: Liza
authors.initials: L
authors.lastname: Pon
authors.forename: Terrance A
authors.initials: TA
authors.lastname: Stadheim
authors.forename: Natarajan
authors.initials: N
authors.lastname: Sethuraman
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D015870:Gene Expression; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D006801:Humans; D018320:Leishmania major / Q000201:enzymology / Q000235:genetics; D060847:Metabolic Engineering; D010843:Pichia / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D016695:Glycosyltransferases