Alkaline-cell lysis through in-line static mixer reactor for the production of plasmid DNA for gene therapy.
A state-of-the-art in-line static mixer reactor (ISMR) was invented to lyse E. coli cells and neutralize the cell lysate continuously and efficiently for the extraction of plasmid DNA. It comprised two connected static dynamic mixers, each 0.01 m in diameter and 0.9 m in length, one for lysis and one for neutralization. Cells were lysed using concentrated alkaline with 1% SDS and the lysate was neutralized at feed rates of cell suspension:lysis solution:neutralization solution of 125:250:125, 250:500:250, and 500:1,000:500 mL/min. Distances for the mixtures to reach color homogeneity were dependent on feed rates. The higher the feed rates the shorter the mixing distances and times. However, complete cell lysis and neutralization were independent of color homogeneity. Lysate viscosity and neutralized floc size decreased and floc density increased, as distances and feed rates increased. High plasmid yields were obtained from both lysis and neutralization at feed rate ratios of 125:250:125 and 250:500:250 mL/min within mixing distances < or =0.6 m. Poor mixing performance and plasmid yield were obtained at a high feed rate of 500:1,000: 500 mL/min when residence and reaction times were less than 2 s and from mixing distances > or =0.6 m at all feed rates due to a longer exposure to strong alkali and shear flow. This invention showed excellent performance with scaleable potential for the commercial manufacture of plasmid DNA.
Chamsart
Saethawat
S
BBERG Bangsaen Biochemical Engineering Research Group, Burapha University, Chon Buri 20131, Thailand. saethawa@buu.ac.th
Karnjanasorn
Tanyawat
T
D004278:DNA, Superhelical / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry*; D015316:Genetic Therapy / Q000295:instrumentation; D010957:Plasmids / Q000737:chemistry*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D012972:Sodium Hydroxide / Q000737:chemistry*
D004278:DNA, Superhelical; D012967:Sodium Dodecyl Sulfate; D012972:Sodium Hydroxide