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.

Plasmid DNA is used as a cloning vector to deliver recombinant genetic information into microorganisms. Since the 1990s, this principle has also been applied for the delivery of therapeutic genes in gene therapy and genetic vaccination. This non-viral gene delivery is afflicted with fewer safety concerns in comparison to viral systems. Processes for the production of high-quality plasmid DNA at multi- and kilogram scale are necessary to meet the needs of clinical trials as well as future therapeutics. Cell disruption, the separation of structurally-related impurities and analytical techniques for process and quality control are the main challenges for bioengineering. This review summarizes the development in these fields over the past recent years.

Plasmid-DNA wird als Klonierungsvektor zum Einbringen rekombinanter Gene in Mikroorganismen genutzt. Seit den 1990er Jahren wird dieses Prinzip auch zur Ubertragung therapeutischer Gene in der Gentherapie und der genetischen Impfung verwendet. Diese Form der nicht viralen Gentherapie stellt eine deutlich risikoarmere Variante im Vergleich zur viralen Gentherapie dar. Zur Deckung des Bedarfs an Plasmid-DNA in klinischen Studien und fur in Zukunft zugelassene Therapeutika sind Produktionsverfahren erforderlich, die eine Herstellung im Multigramm- bis Kilogramm-Masstab moglich machen. Besondere Hersausforderungen werden hier im Bereich des Zellaufschlusses, der Abtrennung strukturell verwandter Verbindungen und der Prozess- und Produktanalytik gestellt. Dieser Ubersichtsbeitrag fasst die Entwicklungen der letzten Jahre zusammen.

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.

The concept of curing diseases at the genetic level was already introduced in the 1970s, but only the evolution of molecular biology and tools for genetic manipulation brought the idea into labs and clinics during the last 16 years. Viral and non-viral vectors and delivery systems were developed to transfer therapeutic genes into the target cells. In the case of non-viral approaches plasmid DNA has become a very promising gene delivery vector because it can easily be genetically manipulated and produced by cultivation of plasmid harbouring Escherichia coli and subsequent downstream processing, thus making production easy in comparison to other gene delivery vectors. Another advantage in using plasmid DNA is the low risk of immunogenic reactions and oncogen activation that can arise while using viral vectors. This review describes the recent development in plasmid manufacturing ranging from bacterial cultivation in batch and fedbatch mode to produce plasmid-bearing E. coli over cell lysis and subsequent purification to storage, application, and process and quality control.

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

The demand for new strategies in downstream processing of biopharmaceutical plasmid DNA has increased in response to the importance of nucleic acids as active pharmaceutical ingredients (API) in gene therapy and genetic vaccination. Led by the problematic usage of animal-derived proteins for producing reagents of clinical applications, we present an opportunity of removing RNA prior to chromatographic steps by using a recombinant RNase Ba (barnase of Bacillus amyloliquefaciens) as an alternative to bovine RNase A. An expression vector for RNase Ba production was constructed enabling periplasmic localization of the recombinant protein. Cultivation of the RNase-producing clone showed stable activity (3.6 kU mL(-1) during stationary phase) throughout the cultivation process. After purification the RNase activity was tested and compared to that of commercially available RNase A. RNase Ba showed no DNase activity even after prolonged incubation with plasmid DNA. Thus, it is a suitable substitute for bovine RNase A in pharmaceutical purification processes.

This chapter contains sections titled: Introduction Gene Therapy DNA Vaccination Manufacturing of Plasmid DNA Bacterial Cultivation Plasmid DNA Purification Innovative Aspects in Plasmid Manufacturing Quality Control of Plasmid DNA Vectors Proteins, Ribonucleic Acid, and Lipopolysaccharides Chromosomal DNA Plasmid Identity Plasmid Topology (Structural Homogeneity) Plasmid Stability during Storage and Application Long-Term Stability of Plasmid DNA Lyophilization for Long-Term Storage Stability during Application Future Developments References

The use of plasmid DNA in gene therapy and genetic vaccination has increased the need for scalable and sustainable production processes. One key challenge for bioprocess engineering is the separation of plasmid DNA from structurally related impurities. Affinity purification procedures allow a highly selective capturing of the target molecule. In this paper, we present the isolation of a his-tagged lac repressor, its non-covalent immobilisation to different matrices and binding of DNA, thus enabling us to screen for combinations of ligands and stationary phases by using a building block principle.

In this paper the influence of ammonium salt concentration on the production of pharmaceutical grade plasmid DNA from unfed high cell density batch culture on synthetic glycerol media is presented. Ammonium chloride in different concentrations (0 mmol dm(-3), 18.7 mmol dm(-3), 37 mmol dm(-3) and 74 mmol dm(-3) supplemented) was used beside sodium glutamate as nitrogen source. Plasmid DNA concentrations of more than 50 mg dm(-3) were obtained with 37 mmol dm(-3) ammonium. The homogeneity of the DNA produced was confirmed by agarose and capillary gel electrophoresis and found to fulfil the quality requirements set for biopharmaceutical plasmid DNA with more than 90% in the supercoiled form. (C) 2003 Society of Chemical Industry.

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.

The transfer of naked DNA is gaining growing acceptance for nonviral gene therapy. Integrity and stability of the DNA used in nonviral gene therapy is known to be decisive for efficacy of gene transfer and transgene expression. Thus, preclinical and clinical studies require the safe storage of DNA preparations to ensure defined quality and conformation. To evaluate the influence of potentially destructive processes on plasmid DNA associated with long-term storage, capillary gel electrophoresis (CGE) analysis of the LacZ-expressing pCMVβ plasmid over a period of 13 months was performed. The CGE analysis revealed that stable storage conditions at −80 °C prevent an increase in open circular (oc) plasmid, preserving the covalently closed circular (ccc) form, which is sought for efficient gene transfer. By contrast, long-term storage of plasmid DNA at 4 °C leads to the rapid decline of the ccc form and the increase of oc and linear DNA molecules. The use of naked DNA stored for 1, 2, or 13 months at −80 °C showed similar in vivo transfer efficiencies by jet-injection. Therefore, analysis of plasmids by CGE allows the reliable determination of integrity and distribution of the topology of the DNA by quantitative means.

Jet-injection has become an applicable technology among other established nonviral delivery systems, such as particle bombardment or in vivo electroporation. The low-volume jet injector employed in this study uses compressed air to inject solutions of 1.5–10 µL containing naked DNA into the desired tissue. The novel design of this prototype makes multiple jet-injections possible. Therefore, repeated jet-injections into one target tissue can be performed easily. This jet-injector hand-held system was used for the direct in vivo gene transfer of plasmid DNA into tumors to achieve efficient expression of reporter genes (β-galactosidase, green fluorescent protein [GFP]) and of therapeutic genes (TNF-α) in different tumor models. The study presented here revealed the key parameters of efficient in vivo jet-injection (jet-injection volume, pressure, jet penetration, DNA stability) to define the optimal conditions for a jet-injection-aided nonviral gene therapy.