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.

journal_name

Glycobiology

journal_title

Glycobiology

authors

Hopkins D,Gomathinayagam S,Rittenhour AM,Du M,Hoyt E,Karaveg K,Mitchell T,Nett JH,Sharkey NJ,Stadheim TA,Li H,Hamilton SR

doi

10.1093/glycob/cwr108

subject

Has Abstract

pub_date

2011-12-01 00:00:00

pages

1616-26

issue

12

eissn

0959-6658

issn

1460-2423

pii

cwr108

journal_volume

21

pub_type

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