Abstract
Monoclonal antibodies, antibody fragments and fusion proteins derived thereof have revolutionized the practice of medicine. Major challenges faced by the biopharmaceutical industry are however high production costs, long processing times and low productivities associated with their production in mammalian cell lines. The yeast Saccharomyces cerevisiae, a well-characterized eukaryotic cell factory possessing the capacity of posttranslational modifications, has been industrially exploited as a secretion host for production of a range of products, including pharmaceuticals. However, due to the incompatible surface glycosylation, few antibody molecules have been functionally expressed in S. cerevisiae. Here, three non-glycosylated antibody fragments from human and the Camelidae family were chosen for expression in a S. cerevisiae strain (HA) previously evolved for high α-amylase secretion. These included the Fab fragment Ranibizumab (Ran), the scFv peptide Pexelizumab (Pex), and a nanobody consisting of a single V-type domain (Nan). Both secretion and biological activities of the antibody fragments were confirmed. In addition, the secretion level of each protein was compared in the wild type (LA) and two evolved strains (HA and MA) with different secretory capacities. We found that the secretion of Ran and Nan was positively correlated with the strains’ secretory capacity, while Pex was most efficiently secreted in the parental strain. To investigate the mechanisms for different secretion abilities in these selected yeast strains for the different antibody fragments, RNA-seq analysis was performed. The results showed that several bioprocesses were significantly enriched for differentially expressed genes when comparing the enriched terms between HA.Nan vs. LA.Nan and HA.Pex vs. LA.Pex, including amino acid metabolism, protein synthesis, cell cycle and others, which indicates that there are unique physiological needs for each antibody fragment secretion.
Highlights
Production of monoclonal antibodies has over the last three decades evolved into an extremely important branch of the biopharmaceutical industry
Two anti-MUC1 VhHs were successfully expressed in Pichia pastoris for the first time, and titers in the range of 10–15 mg/L were obtained after a series of optimizations [14]
Of the 2A peptides identified to date, four with varied cleavage efficiency have been commonly used in biomedical research: F2A from foot-andmouth disease virus, E2A from equine rhinitis A virus, P2A from porcine teschovirus-1 and T2A from Thosea asigna virus [50]
Summary
Production of monoclonal antibodies (mAbs) has over the last three decades evolved into an extremely important branch of the biopharmaceutical industry. Recombinant DNA technology opened the possibility of expressing antibody fragment genes in heterologous host organisms that are easy to manipulate and cultivate. Through engineering of E. coli, several grams per liter of the recombinant antibody fragments scFv and Fab can be achieved in fermenters [13]. Compared with the bacterial expression systems, yeasts prove to be more desirable hosts for industrialscale production of recombinant proteins due to their ability to perform post-translational modifications. Their high robustness and tolerance towards harsh fermentation conditions, which are key factors in bioprocessing and scale-up, are additional advantages. The absence of glycosylation sites on many antibody fragments circumvents this problem, further adding to the good prospect for yeast as a platform for production of this group of pharmaceuticals
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