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.
Fluorescence Signal Good Manufacturing Practice Transient Gene Expression Buffer Composition Linear Standard Curve
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Akinc A, Thomas M, Klibanov AM, Langer R (2005) Exploring polyethylenimine-mediated DNA transfection and the proton sponge hypothesis. J Gene Med 7: 657–663PubMedCrossRefGoogle Scholar
Backliwal G, Hildinger M, Hasija V, Wurm FM (2008) High-density transfection with HEK-293 cells allows doubling of transient titers and removes need for a priori DNA complex formation with PEI. Biotechnol Bioeng 99: 721–727PubMedCrossRefGoogle Scholar
Bertschinger M, Burki C, Backliwal G, Hacker DL, Jordan M, Wurm FM (2006) Polyethylenimine-based quality control assay for plasmid DNA. Anal Biochem 356: 309–311PubMedCrossRefGoogle Scholar
Choosakoonkriang S, Lobo BA, Koe GS, Koe JG, Middaugh CR (2003) Biophysical characterization of PEI/DNA complexes. J Pharm Sci 92: 1710–1722PubMedCrossRefGoogle Scholar
Godbey WT, Wu KK, Mikos AG (1999) Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery. Proc Natl Acad Sci USA 96: 5177–5181PubMedCrossRefGoogle Scholar
Gratama JW (1998) Flow cytometric quantitation of immunofluorescence intensity: Problems and perspectives. Cytometry 33: 166–178PubMedCrossRefGoogle Scholar
Long GL, Winefordner JD (1983) Limit of detection. A closer look at the IUPAC definition. Anal Chem 55: 712A–724ACrossRefGoogle Scholar
Seib FP, Jones AT, Duncan R (2007) Comparison of the endocytic properties of linear and branched PEIs, and cationic PAMAM dendrimers in B16f10 melanoma cells. J Control Release 117: 291–300PubMedCrossRefGoogle Scholar