On the stability of plasmid DNA vectors during cell culture and purification

Abstract

Gene therapy and DNA vaccination applications have increased the demand for highly purified plasmid DNA (pDNA) in the last years. One of the main problems related to the scale-up of pDNA purification is the degradation of the supercoiled (sc) isoforms during cell culture and multi-stage purification. In this work, a systematic study of the stability of two model plasmids (3,697 and 6,050 bp) during a mid-scale production process, which includes fermentation, alkaline lysis, isopropanol and ammonium sulphate precipitation and hydrophobic interaction chromatography, was performed. Results indicate that by extending cell culture (up to 26 h) and cell lysis (up to 2 h) it is possible to significantly reduce the amounts of RNA, without significantly compromising the yields of the sc pDNA isoform, a feature that could be conveniently exploited for downstream processing purposes. The stability of pDNA upon storage of E. coli pellets at different temperatures indicates that, differently from RNA, pDNA is remarkably stable when stored in cell pellets (>3 weeks at 4 degrees C, >12 weeks at -20 degrees C) prior to processing. With alkaline lysates, however, storage at -20 degrees C is mandatory to avoid sc pDNA degradation within the first 8 weeks. Furthermore, the subsequent purification steps could be carried out at room temperature without significant pDNA degradation. Since the unit operations and process conditions studied in this work are similar to those generally used for plasmid DNA production, the results presented here may contribute to improve the current knowledge on plasmid stability and process optimization.

Development of a recombinant fusion protein based on the dynein light chain LC8 for non-viral gene delivery

“…The success of the reporter gene replacement was confirmed by the expression of the reporter enzyme after transfection of HeLa cells, as described in this work. Purification of the pVAX1-Luc plasmid used in all transfection studies was performed as described by Freitas and co-workers [28].…”

Targeted Gene Therapy for the Treatment of Heart Failure

“…21,22 A major advantage is that there are no size limitations, as with viral vectors, which allows the utilization of large transgenes or necessary elements in the expression cassette. 18,23 Additionally, naked DNA can be produced in large amounts using standard techniques without any significant increase in cost, although significant improvements are constantly investigated, 24 is very stable and can be stored for prolonged periods of time without any effect on quality. The method of administration for naked DNA is direct injection, because DNA cannot cross the vascular cell barrier via infusion.…”

Uptake of Foreign Nucleic Acids in Kidney Tubular Epithelial Cells Deficient in Proapoptotic Endonucleases

“…E xtracellular DNA uptake occurs during normal and cancer tissue growth (Bergsmedh et al, 2006;Yan et al, 2006) and during viral and bacterial infections (Chu et al, 2006;Metifiot et al, 2007), and is routinely used in genetic manipulations and experimental animals (Tanswell et al, 1998;Glasspool-Malone et al, 2002;Freitas et al, 2007). The entry of foreign DNA (fDNA) is harmful to the host cell (Li et al, 1999), causing DNA-dependent cell death, whereas DNase treatment before transfection prevented cell death (Stacey et al, 1993).…”

“…Despite attempts to protect the fDNA by modifications, lipid or viral packaging, increased rate of DNA delivery, or by precise targeting to a tissue, our knowledge about the protection against the cellular defense system remained insufficient (Tanswell et al, 1998;Glasspool-Malone et al, 2002;Freitas et al, 2007). Endocytosis of DNA normally leads to its lysosomal delivery, with DNases playing major role in degrading fDNA (Torchilin, 2006).…”