N-Glycosylation Humanization for Production of Therapeutic Recombinant Glycoproteins in Saccharomyces cerevisiae

Abstract

The production of therapeutic recombinant glycoproteins deals with three main issues: cost, production capacities, and glycosylation. Nowadays, such proteins are expressed in various complex expression systems (CHO, bacteria, etc.); the processes related to those production hosts are time consuming and expensive, or the question of posttranslational modifications (as glycosylation) control is still unresolved. There is a need to find an alternative approach, while maintaining high quality level: the new system must be able to add complex N-glycan structures to proteins of interest. Developed in several strains of Saccharomyces cerevisiae, GlycodExpress™ is an innovative technology that allows production of therapeutic recombinant glycoproteins with humanized and homogeneous N-glycan moieties. We show how to delete mannosyltransferases involved in host N-glycosylation to obtain more than 90% of homogeneity in glycan structures. The methodology developed to select the optimal fusion between a heterologous glycosyl-enzyme and a localization sequences is also presented. Finally, the screening of the best producing strain is illustrated.

Characterization of Therapeutic Antibodies and Related Products

“…Upstream from the market production, smaller amounts of mAbs can be produced by transient gene expression in HEK-293E cells in a reproducible manner resulting in glycoforms similar to those observed in human IgGs . Therapeutic antibodies may also be produced in alternative systems such as Pichia pastoris , Saccharomyces cerevisiae , and lepidopteran cells . N-Glycosylations of IgGs are asymmetrical and may even result in hemiglycosylated IgG, i.e., antibodies with one glycosylated HC and another aglycosylated …”

N-hypermannose glycosylation disruption enhances recombinant protein production by regulating secretory pathway and cell wall integrity in Saccharomyces cerevisiae

“…The glycosylation pathway modifications have been studied extensively in Pichia pastoris 5 . In S. cerevisiae , the expression of heterologous proteins often results in a hypermannose glycan structure that may result in less activity and changes to their immunogenicity 6 7 8 9 . Therefore, reducing hypermannose glycans may be one of the key steps for the recombinant protein production.…”

The synthesis of recombinant membrane proteins in yeast for structural studies

“…Complementing this, Dharmacon sells the Yeast Tet-Promoters Hughes Collection (yTHC) with 800 essential yeast genes under control of a tetracycline-regulated promoter that permits experimental regulation of essential genes. A number of specifically-engineered S. cerevisiae strains also exist including those with ''humanized'' sterol (see section 4.3) and glycosylation pathways [44]. Notably, protease-deficient strains are a consistently-popular choice in membrane protein structural biology projects ( Table 2).…”