Abstract
Chinese hamster ovary (CHO) cells are widely used as cell factories for the production of biopharmaceuticals. In contrast to the highly optimized production processes for monoclonal antibody (mAb)-based biopharmaceuticals, improving productivity of non-mAb therapeutic glycoproteins is more likely to reduce production costs significantly. The aim of this study was to establish a versatile target gene screening platform for improving productivity for primarily non-mAb glycoproteins with complete interchangeability of model proteins and target genes using transient expression. The platform consists of four techniques compatible with 96-well microplates: lipid-based transient transfection, cell cultivation in microplates, cell counting and antibody-independent product titer determination based on split-GFP complementation. We were able to demonstrate growth profiles and volumetric productivity of CHO cells in 96-half-deepwell microplates comparable with those obtained in shake flasks. In addition, we demonstrate that split-GFP complementation can be used to accurately measure relative titers of therapeutic glycoproteins. Using this platform, we were able to detect target gene-specific increase in titer and specific productivity of two non-mAb glycoproteins. In conclusion, the platform provides a novel miniaturized and parallelisable solution for screening target genes and holds the potential to unravel genes that can enhance the secretory capacity of CHO cells.
Highlights
Chinese hamster ovary (CHO) cells are widely used as cell factories for the production of biopharmaceuticals
Hoechst stain was used to identify the total number of cells and propidium iodide stain was used to identify dead cells
viable cell density (VCD) measurements of the 96-well-based assay were consistent with results obtained with the commercially available acridine orange- and DAPI-based NucleoCounter NC-200 Cell Counter (Fig. 2a,b)
Summary
Chinese hamster ovary (CHO) cells are widely used as cell factories for the production of biopharmaceuticals. In contrast to the highly optimized production processes for monoclonal antibody (mAb)-based biopharmaceuticals, improving productivity of non-mAb therapeutic glycoproteins is more likely to reduce production costs significantly. We demonstrate that split-GFP complementation can be used to accurately measure relative titers of therapeutic glycoproteins. Using this platform, we were able to detect target gene-specific increase in titer and specific productivity of two non-mAb glycoproteins. A transient transfection-based overexpression screening platform has the potential to address these issues, because it enables complete interchangeability of which genes (hereafter referred to as target genes) and model therapeutic glycoproteins (hereafter referred to as model proteins) to express. The S11_M3-tag is less likely to perturb folding processes and solubility than previous, larger versions of the split-GFP tag[13,14]
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