The impact of fluid-dynamic-generated stresses on chDNA and pDNA stability during alkaline cell lysis for gene therapy products.
Extensive tests have been carried out to assess the impact of fluid-dynamic-generated stress during alkaline lysis of Escherichia coli cells (host strain DH1 containing the plasmid pTX 0161) to produce a plasmid DNA (pDNA) solution for gene therapy. Both a concentric cylinder rheometer and two stirred reactors have been used, and both the alkaline addition and neutralization stages of lysis have been studied. Using a range of shear rates in the rheometer, stirrer speeds in the reactors, and different periods of exposure, their impact on chromosomal DNA (chDNA) and pDNA was assessed using agarose gel electrophoresis, a Qiagen Maxiprep with a polymerase chain reaction (PCR) assay, and a Qiagen Miniprep purification with a UV spectrophotometer. Comparison has been made with unstressed material subjected to similar holding times. These tests essentially show that under all these conditions, <2% chDNA was present in the pDNA solution, the pDNA itself was not fragmented, and a yield of 1 mg/g cell was obtained. These results, together with studies of rheological properties, have led to the design of a 60-L, stirred lysis reactor and the production of high-quality pDNA solution with <1% chDNA after further purification.
Chamsart
S
S
Centre for Bioprocess Engineering, School of Chemical Engineering, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
Patel
H
H
Hanak
J A
JA
Hitchcock
A G
AG
Nienow
A W
AW
D019149:Bioreactors; D001709:Biotechnology; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D006863:Hydrogen-Ion Concentration; D010957:Plasmids / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012996:Solutions; D013314:Stress, Mechanical; D013997:Time Factors
D004269:DNA, Bacterial; D012996:Solutions