Elimination of β-mannose glycan structures in Pichia pastoris

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....

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Bibliographic Details
Published in:Glycobiology (Oxford) 2011-12, Vol.21 (12), p.1616-1626
Main Authors: Hopkins, Daniel, Gomathinayagam, Sujatha, Rittenhour, Alissa M, Du, Min, Hoyt, Erik, Karaveg, Khanita, Mitchell, Teresa, Nett, Juergen H, Sharkey, Nathan J, Stadheim, Terrance A, Li, Huijuan, Hamilton, Stephen R
Format: Article
Language:English
Subjects:
Carbohydrate Conformation
Cross Reactions
Erythropoietin - chemistry
Erythropoietin - genetics
Erythropoietin - isolation & purification
Erythropoietin - metabolism
Humans
Mannose - chemistry
Mannose - metabolism
Mannosyltransferases - genetics
Mannosyltransferases - metabolism
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase - metabolism
Pichia - chemistry
Pichia - enzymology
Pichia - genetics
Polysaccharides - chemistry
Polysaccharides - metabolism
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - isolation & purification
Recombinant Proteins - metabolism
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Summary: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.
ISSN:1460-2423
DOI:10.1093/glycob/cwr108