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Animal‐free production of ccc‐supercoiled plasmids for research and clinical applications
Author(s) -
Schleef Martin,
Schmidt Torsten
Publication year - 2004
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.511
Subject(s) - plasmid , plasmid preparation , dna , capillary electrophoresis , chemistry , biology , chromatography , biochemistry , pbr322
Abstract The topological structure of plasmid DNA can be characterized by capillary gel electrophoresis (CGE analysis)—an important tool for quality control and stability assessments in DNA storage or application. Hence, a large‐scale manufacturing process was developed that allows the removal of undesired open circular (oc) or linear plasmid topologies, bacterial genomic DNA, RNA, proteins as well as lipopolysaccharides (endotoxins) and results in obtaining supercoiled (covalently closed circular, ccc) plasmid DNA in a pure form without using any animal‐derived substances. Using CGE, the development and in‐line monitoring for pharmaceutical plasmid production starting from fermentation control throughout the whole manufacturing process including the formulated and filled product can be performed the first time in a way conforming to good manufacturing practices (GMP). Plasmid stability data were obtained from analysis of shear effects influencing the plasmid quality in DNA drug delivery formulation and application (e.g. gene gun or jet injection). The physical stability of plasmid DNA is for the first time evaluated in DNA storage experiments on the level of different plasmid forms. Copyright © 2004 John Wiley & Sons, Ltd.