Expression of antibody fragments in Saccharomyces cerevisiae strains evolved for enhanced protein secretion.

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 post-translational 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.

Identification of multiple serine to asparagine sequence variation sites in an intended copy product of LUCENTIS® by mass spectrometry.

Patent expiration of first-generation biologics and the high cost of innovative biologics are 2 drivers for the development of biosimilar products. There are, however, technical challenges to the production of exact copies of such large molecules. In this study, we performed a head-to-head comparison between the originator anti-VEGF-A Fab product LUCENTIS® (ranibizumab) and an intended copy product using an integrated analytical approach. While no differences could be observed using size-exclusion chromatography, capillary electrophoresis-sodium dodecyl sulfate and potency assays, different acidic peaks were identified with cation ion exchange chromatography and capillary zone electrophoresis. Further investigation of the intact Fab, subunits and primary sequence with mass spectrometry demonstrated the presence of a modified light chain variant in the intended copy product batches. This variant was characterized with a mass increase of 27.01 Da compared to the originator sequence and its abundance was estimated in the range of 6-9% of the intended copy product light chain. MS/MS spectra interrogation confirmed that this modification relates to a serine to asparagine sequence variant found in the intended copy product light chain. We demonstrated that the integration of high-resolution and sensitive orthogonal technologies was beneficial to assess the similarity of an originator and an intended copy product.

Functional Characterization of AAV-Expressed Recombinant Anti-VEGF Single-Chain Variable Fragments In Vitro.

PURPOSE: Most retinal neovascular disorders are caused by upregulation of vascular endothelial growth factor (VEGF) expression. These disorders are treated with repeated injections of anti-VEGF molecules, which may have severe side effects. The expression of anti-VEGF molecules by the retina itself in a controlled manner following adeno-associated viral (AAV) gene transfer could be a replacement of this therapy. METHODS: The open reading frames (orf) of the light and the heavy chain of ranibizumab were cloned into an expression plasmid separated by an internal ribosomal entry site (IRES). The construct was mutated to generate ranibizumab single-chain variable fragments (scFv). Expression was verified by western blotting and the concentrations were measured with a custom-made ranibizumab ELISA. Biological activity, VEGF-binding properties, and the doxycycline-dependent induction of anti-VEGF expression were tested. An AAV2/5 vector was generated containing the optimal variant Ra02. RESULTS: Ra01-Ra05 molecules were detected in the cell culture medium. While the VEGF-binding affinity was significantly lower for Ra01 and Ra02 compared to Lucentis(®), the inhibition of cell migration was comparable and the maximum inhibition of Ra01 and Ra02 was reached at lower doses. The expression of Ra01 and Ra02 was shown to be regulable with the TetOn-system(®) as plasmid (Ra01, Ra02) and AAV vector construct (Ra02). CONCLUSION: Ra01 and Ra02 can be produced in eukaryotic cells after AAV-mediated gene transfer in a regulable manner in vitro and display comparable biological activity as Lucentis. These results are the basis for in vivo studies in human VEGF-overexpressing mice, a model for human neovascular disorders.