The separation of structurally related impurities from pharmaceutical plasmid DNA by highly scalable purification techniques is a challenge for biochemical engineering. Next to RNA, proteins, and lipopolysaccharides, the chromosomal DNA of the plasmid replicating host has to be removed. Here, we describe the application of reverse micellar extraction for the separation of chromosomal from plasmid DNA. By applying different procedures for alkaline lysis, bacterial lysates with different amounts of chromosomal DNA were generated. A reverse micellar extraction step enabled us to deplete the concentration of this impurity below the required level of 50 mg g(-1) of plasmid DNA with almost complete plasmid recovery.

AbstractDie Verteilung von RNA und Plasmid‐DNA wurde in einem inversmizellaren Zweiphasensystem bestehend aus Trioctylmethylammoniumchlorid (TOMAC) und Isooctan untersucht. Die Auswirkung verschiedener Salze und Alkohole auf den Transfer der Nukleinsäuren wurde vor allem unter dem Gesichtspunkt einer hohen Wiederfindung der Plasmid‐DNA nach der Rückextraktion betrachtet. Systeme, die eine Trennung von Plasmid‐DNA und RNA ermöglichen, wurden eingesetzt, um RNA aus bakteriellem Klarlysat zu entfernen.

Plasmid DNA as an active pharmaceutical ingredient (API) is gaining more and more importance. For the production of multigram quantities of this substance robust and scalable processes comprising several purification steps have to be designed. One main challenge is the initial separation of plasmid DNA and RNA in such a purification scheme. In this study we investigated the distribution of plasmid DNA and RNA in reverse micellar two-phase systems which is considered to be the basis for the development of an extractive purification step that can easily be integrated into common processes. For this purpose the distribution of the 4.6 kb plasmid pUT649 and Escherichia coli RNA in systems comprising isooctane, ethylhexanol, and the surfactant methyltrioctylammoniumchloride (TOMAC) under the influence of different salts was studied. Anion concentrations at which the partitioning behaviour for nucleic acids inverted (inversion point) were identified. Systems capable of separating RNA from plasmid DNA were further analysed and applied to extract RNA from plasmid DNA out of a preconditioned cleared lysate. The capability of reverse micellar systems for plasmid form separation was also shown by capillary and agarose gel electrophoresis.

Chemie Ingenieur TechnikVolume 78, Issue 9 p. 1389-1389 PosterFree Access Extraktion von Nucleinsäuren mittels inversmizellarer Zweiphasensysteme N. Streitner Dipl. Ing., N. Streitner Dipl. Ing. nst@fermtech.techfak.uni-bielefeld.de Universität Bielefeld, Technische Fakultät, Lehrstuhl für Fermentationstechnik, Universitätsstraße 25, D-33615 BielefeldSearch for more papers by this authorC. Voß Dr., C. Voß Dr. Universität Bielefeld, Technische Fakultät, Lehrstuhl für Fermentationstechnik, Universitätsstraße 25, D-33615 BielefeldSearch for more papers by this authorE. Flaschel Prof. Dr., E. Flaschel Prof. Dr. Universität Bielefeld, Technische Fakultät, Lehrstuhl für Fermentationstechnik, Universitätsstraße 25, D-33615 BielefeldSearch for more papers by this author N. Streitner Dipl. Ing., N. Streitner Dipl. Ing. nst@fermtech.techfak.uni-bielefeld.de Universität Bielefeld, Technische Fakultät, Lehrstuhl für Fermentationstechnik, Universitätsstraße 25, D-33615 BielefeldSearch for more papers by this authorC. Voß Dr., C. Voß Dr. Universität Bielefeld, Technische Fakultät, Lehrstuhl für Fermentationstechnik, Universitätsstraße 25, D-33615 BielefeldSearch for more papers by this authorE. Flaschel Prof. Dr., E. Flaschel Prof. Dr. Universität Bielefeld, Technische Fakultät, Lehrstuhl für Fermentationstechnik, Universitätsstraße 25, D-33615 BielefeldSearch for more papers by this author First published: 06 September 2006 https://doi.org/10.1002/cite.200650205AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume78, Issue9Special Issue: GVC/DECHEMA‐Jahrestagungen 2006 mit 24. DECHEMA‐Jahrestagung der BiotechnologenSeptember, 2006Pages 1389-1389 RelatedInformation