Production of supercoiled multimeric plasmid DNA for biopharmaceutical application.
Production of nucleic acids as an active pharmaceutical ingredient (API) in gene therapy and genetic vaccination is gaining more and more importance. Non-viral vectors like plasmid DNA are currently investigated in various clinical trials. Supercoiled multimeric plasmids are of particular interest for pharmaceutical purpose because they contain multiple copies of a therapeutic gene and can therefore be more efficient vectors. A process for the preparation of Escherichia coli strains replicating dimers, trimers, and tetramers of a 4.6 kb plasmid is presented. Cultivation of these clones on semi-defined glycerol medium in a 7 l bioreactor shows structural stability of dimers and trimers during the whole cultivation process. Plasmid concentrations and selectivities are compared to the corresponding cultivation with the plasmid monomer. Cultivation of the tetramer replicating strain shows a disintegration of the plasmid multimer and reconstitution of the monomer and smaller multimers.
Voss
Carsten
C
Chair of Fermentation Engineering, Faculty of Technology, University of Bielefeld, P.O. 100 131, Bielefeld D-33501, Germany. cvo@fermtech.techfak.uni-bielefeld.de
Schmidt
Torsten
T
Schleef
Martin
M
Friehs
Karl
K
Flaschel
Erwin
E
D001702:Biopharmaceutics; D019149:Bioreactors; D001709:Biotechnology / Q000379:methods; D003001:Cloning, Molecular; D003470:Culture Media; D004261:DNA Replication; D004278:DNA, Superhelical / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D005822:Genetic Vectors; D010957:Plasmids
D003470:Culture Media; D004278:DNA, Superhelical