title: Development of a phenyl membrane chromatography-based process yielding pharmaceutical grade plasmid deoxyribonucleic acid for mammalian cells transfection.
abstract: Production of plasmid DNA pharmaceuticals requires fast, robust and cost effective methodologies able to deliver high amounts of the target molecule in short periods of time. Membrane adsorbers can be tailored and operated to suit such criteria. This study focuses on the impact of pDNA samples produced by a membrane chromatography-based purification methodology on the transfection efficiency of CHO cells. Chromatographies were performed with 5mL of plasmid-containing clarified bacterial lysate each on a Sartorius® Phenyl 3mL spiral cartridge using a bind and elute mode to purify the GFP expressing pVAX1/GFP model plasmid. The developed methodology could deliver up to 285μg pDNA samples per run that were virtually RNA free (over 99% removal) and chromatographic step yields of 85%. The purified samples had a reduced content of OC pDNA (∼15% less in average). Additionally, robustness of the process was assessed up to nine chromatographic runs without noticing any relevant loss in chromatographic performance and transfection capabilities. The increase of productivity was also studied by increasing the flow rate 5 fold-a maximum productivity of 100μg pDNA/(hmL-BV) was achieved. The pDNA samples produced led to transfection efficiencies that were comparable among all experiments-72% and within 4% relative standard deviation when samples were produced using a lower throughput. Transfection efficiencies obtained by the membrane process were comparable to a combined HIC/SEC bead-based purification process, with values ranging within 74-113% of the values obtained from the latter.
authors.affiliation: IBB - Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
authors.forename: Luís
authors.initials: L
authors.lastname: Raiado-Pereira
authors.affiliation: IBB - Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
authors.forename: Jonathan
authors.initials: J
authors.lastname: De La Vega
authors.affiliation: IBB - Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
authors.forename: D Miguel F
authors.initials: DM
authors.lastname: Prazeres
authors.affiliation: IBB - Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal. Electronic address: marilia.mateus@tecnico.ulisboa.pt.
authors.forename: Marília
authors.initials: M
authors.lastname: Mateus
mesh_terms: D000327:Adsorption; D000818:Animals; D016466:CHO Cells; D002845:Chromatography / Q000379:methods; D003412:Cricetulus; D004247:DNA / Q000302:isolation & purification*; D004926:Escherichia coli / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D008567:Membranes, Artificial; D010957:Plasmids; D014162:Transfection
chemical_list: D008567:Membranes, Artificial; D004247:DNA
keywords: Biopharmaceuticals DSP; CHO cell transfection; Hydrophobic interaction chromatography; Membrane adsorbers; Plasmid DNA; Process chromatography of plasmids