On the stability of plasmid DNA vectors during cell culture and purification.
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.
Freitas
S S
SS
IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, 1049-001 Lisboa, Portugal.
Azzoni
A R
AR
Santos
J A L
JA
Monteiro
G A
GA
Prazeres
D M F
DM
D019840:2-Propanol / Q000737:chemistry; D000468:Alkalies / Q000737:chemistry; D018929:Cell Culture Techniques / Q000379:methods*; D004278:DNA, Superhelical / Q000737:chemistry* / Q000302:isolation & purification; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000254:growth & development*; D005822:Genetic Vectors / Q000737:chemistry* / Q000302:isolation & purification; D010957:Plasmids / Q000737:chemistry* / Q000302:isolation & purification; D012492:Salts / Q000737:chemistry
D000468:Alkalies; D004278:DNA, Superhelical; D012492:Salts; D019840:2-Propanol