TY - JOUR
T1 - The impact of fluid-dynamic-generated stresses on chDNA and pDNA stability during alkaline cell lysis for gene therapy products
AU - Chamsart, Saedthawat
AU - [No Value], [No Value]
AU - Hanak, JAJ
AU - Hitchcock, AG
AU - Nienow, Alvin
PY - 2001/11/20
Y1 - 2001/11/20
N2 - 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 dierent 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,
AB - 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 dierent 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,
KW - alkaline lysis
KW - chromosomal DNA
KW - plasmid DNA
KW - fluid dynamic stresses
KW - stability
UR - http://www.scopus.com/inward/record.url?scp=0035923311&partnerID=8YFLogxK
U2 - 10.1002/bit.10046
DO - 10.1002/bit.10046
M3 - Article
SN - 1097-0290
VL - 75
SP - 387
EP - 392
JO - Biotechnology and Bioengineering
JF - Biotechnology and Bioengineering
ER -