title: Elimination of β-mannose glycan structures in Pichia pastoris.
abstract: The methylotrophic yeast, Pichia pastoris, is an important organism used for the production of therapeutic proteins. However, the presence of fungal-like glycans, such as those containing β-mannose (Man) linkages, can elicit an immune response or bind to Man receptors, thus reducing their efficacy. Recent studies have confirmed that P. pastoris has four genes from the β-mannosyl transferase (BMT) family and that Bmt2p is responsible for the majority of β-Man linkages on glycans. While expressing recombinant human erythropoietin (rhEPO) in a developmental glycoengineered strain devoid of BMT2 gene expression, cross-reactivity was observed with an antibody raised against host cell antigens. Treatment of the rhEPO with protein N-glycosidase F eliminated cross-reactivity, indicating that the antigen was associated with the glycan. Thorough analysis of the glycan profile of rhEPO demonstrated the presence of low amounts of α-1,2-mannosidase resistant high-Man glycoforms. In an attempt to eliminate the α-mannosidase resistant glycoforms, we used a systemic approach to genetically knock-out the remaining members of the BMT family culminating in a quadruple bmt2,4,1,3 knock-out strain. Data presented here conclude that the additive elimination of Bmt2p, Bmt3p and Bmt1p activities are required for total abolition of β-Man-associated glycans and their related antigenicity. Taken together, the elimination of β-Man containing glycoforms represents an important step forward for the Pichia production platform as a suitable system for the production of therapeutic glycoproteins.
authors.affiliation: GlycoFi Inc., A wholly-Owned Subsidiary of Merck & Co. Inc., 21 Lafayette street, Suite 200, Lebanon, NH 03766, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Hopkins
authors.forename: Sujatha
authors.initials: S
authors.lastname: Gomathinayagam
authors.forename: Alissa M
authors.initials: AM
authors.lastname: Rittenhour
authors.forename: Min
authors.initials: M
authors.lastname: Du
authors.forename: Erik
authors.initials: E
authors.lastname: Hoyt
authors.forename: Khanita
authors.initials: K
authors.lastname: Karaveg
authors.forename: Teresa
authors.initials: T
authors.lastname: Mitchell
authors.forename: Juergen H
authors.initials: JH
authors.lastname: Nett
authors.forename: Nathan J
authors.initials: NJ
authors.lastname: Sharkey
authors.forename: Terrance A
authors.initials: TA
authors.lastname: Stadheim
authors.forename: Huijuan
authors.initials: H
authors.lastname: Li
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Hamilton
mesh_terms: D002236:Carbohydrate Conformation; D003429:Cross Reactions; D004921:Erythropoietin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D008358:Mannose / Q000737:chemistry* / Q000378:metabolism; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D010843:Pichia / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011134:Polysaccharides; D011994:Recombinant Proteins; D004921:Erythropoietin; D008364:Mannosyltransferases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D008358:Mannose