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

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.

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 forpharmaceutical 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 (C) 2004 John Wiley Sons, Ltd.

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.

The development of innovative therapy forms as, e.g., cell and gene therapy or vaccination with DNA, has generated new challenges in process development and quality assurance for the pharmaceutical manufacturing of DNA drugs. Vaccination with DNA has shown first clinical success in human and veterinary medicine. This study summarizes the mode of action of DNA vaccines and focuses on the relevant aspects of manufacturing such APIs (active pharmaceutical ingredients), especially with respect to quality assurance.

ChemBioChemVolume 2, Issue 11 p. 853-853 Book ReviewFree Access Book Review: Capillary Electrophoresis of Nucleic Acids Volumes I and II Edited by Keith R. Mitchelson and Jing Cheng Torsten Schmidt, Torsten Schmidt PlasmidFactory GmbH & Co. KG Bielefeld (Germany)Search for more papers by this author Torsten Schmidt, Torsten Schmidt PlasmidFactory GmbH & Co. KG Bielefeld (Germany)Search for more papers by this author First published: 30 October 2001 https://doi.org/10.1002/1439-7633(20011105)2:11<853::AID-CBIC2222853>3.0.CO;2-0AboutPDF 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. Volume2, Issue11November 5, 2001Pages 853-853 RelatedInformation

With pharmaceutical-grade plasmid DNA, an adequate homogeneity of the final product is achieved when more than 90% of the molecules exist as the supercoiled covalently closed circular (ccc) form. This is the most compact form where the circular and covalently closed DNA helix is interwoven in itself, like a twisted rubber band. If one of the DNA strands is broken, the circular molecule relaxes under loss of coiling. This relaxed structure is called the open circular (oc) or nicked form. These topological plasmid structures may exist as different sizes, such as monomers and dimers, creating additional heterogeneity. Finally, linear plasmid structures are generated when both strands are cleaved at the same position.

Plasmids may appear in different forms: circular with different degrees of coiling, partially cleaved or linear, and multimeric as concatamers or catenates. Capillary gel electrophoresis (CGE) of plasmid samples allows the determination of plasmid form distribution. Monomeric and dimeric plasmid DNA forms were separated by both CGE and agarose gel electrophoresis (AGE). The pattern of isoform bands from AGE was compared to the corresponding peak pattern from CGE, and differences in the relative mobility of the plasmid forms between the two methods were found. The comparison of AGE and CGE allows the assignment of AGE bands to CGE peaks. Additionally, the different isoforms can now be quantified by CGE. Routine plasmid form analysis by CGE may be automated, allowing easy, fast, and highly reliable quantification. CGE also offers high resolution and the amount of DNA required is very low. Therefore this method is very useful for the analysis of therapeutics based on plasmid DNA during their production, isolation, and formulation.

Plasmid copy number, the number of expression vectors per host cell, is a key variable in recombinant microbial cultivation. Therefore, it would be very helpful, if the plasmid copy number could be determined during the operating process period. A rapid quantification of this important process variable would even open the possibility of its use in process control. However, current assays like gel electrophoresis, CsCl-gradient centrifugation, HPLC and other methods are time consuming and difficult to quantify. Indirect methods, like the correlation of copy number with e.g. the activity of an enzyme, coded on the plasmid, are prone to errors due to the production kinetics, turnover rate and protein denaturation. Here, a method is presented, which enables the plasmid copy number to be determined in less than 30 min. This novel procedure based on plasmid isolation by means of a commercial DNA-isolation kit and quantification by capillary electrophoresis, should allow the copy number to be used in process control.