| Popis: |
Transient gene expression (TGE) allows production of virtually any recombinant protein (r-protein) in mammalian cells. Its flexibility, speed, scalability, and cost-effectiveness have been widely demonstrated. However, good manufacturing practices (GMP) have not been established for the production of r-proteins by TGE. In this study, a method was developed for the detection and quantification of polyethylenimine (PEI), the DNA delivery agent in TGE. Currently, there are no established methods to track this polymer during r-protein production and purification. Linear 25 kDa PEI was labelled with fluorescein, and the modified PEI was characterized by NMR and UV/VIS spectroscopy. The optimal conditions for an accurate measurement of PEI by fluorescence were defined, and the limit of detection and quantification were determined. Importantly, the labeling of PEI did not alter its capacity to form polyplexes with plasmid DNA and to efficiently transfect HEK-293 cells in suspension. The assay we developed is expected to be an essential tool for the establishment of GMP protocols for the production of r-proteins by TGE. |
