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Cai Y.,VGX Pharmaceuticals Inc. | Rodriguez S.,VGXI Inc. | Rameswaran R.,VGXI Inc. | Draghia-Akli R.,VGX Pharmaceuticals Inc. | And 2 more authors.
Vaccine | Year: 2010

The increased use of plasmid-based vaccines to replace their more challenging viral counterparts has increased the demand for high purity and high concentration plasmids. Here we report the production of plasmids encoding different transgenes for DNA vaccine candidates at gram scale with an integrated process consisting of batch fermentation and limited steps of purification. Plasmid products encoding for eight smallpox antigens that were combined into a bioterrorism DNA vaccine exhibited high purity with undetectable RNA, protein and endotoxin, concentration of up to 13.6 mg/mL and supercoiled percentage of 94.5 ± 1.1% after storage at -80 °C for over 1 year. The process has been scaled up for the cGMP manufacture of pharmaceutical-grade human papillomavirus and influenza DNA vaccines up to a 50 g scale, also demonstrating high purity and high concentration. © 2009 Elsevier Ltd. All rights reserved.

Nelson J.,VGXI Inc. | Rodriguez S.,VGXI Inc. | Finlayson N.,Coridon Pty Ltd. | Williams J.,Nature Technology Corporation | Carnes A.,Nature Technology Corporation
Human Vaccines and Immunotherapeutics | Year: 2013

Two DNA vaccine plasmids encoding Herpes simplex virus type 2 (HSV-2) glycoprotein D, NTC8485-02-gD2and NTC8485-02-UgD2tr, were produced at large scale under current good manufacturing practice (cGMP) for use in a Phase I human clinical trial. These DNA vaccines incorporate the regulatory agency compliant, minimal, antibiotic-free (AF) NTC8485 mammalian expression vector. Plasmid yields of > 1 g/L were achieved using the HyperGRO™ fed-batch fermentation process, with successful scale up from 10 L process development scale to 320 L culture volume for cGMP production. The DNA vaccines were purified using a low residence time, high shear lysis process and AIRMIX™ technology, followed by chromatographic purification. This combination of optimized plasmid vector, high yield upstream production, and efficient downstream purification resulted in purified HSV-2 DNA vaccines with > 99% total supercoiled plasmid, ≤ 0.2% RNA, ≤ 0.1% host cell genomic DNA, and ≤ 0.1 endotoxin units per mg. © 2013 Landes Bioscience.

Bazzani R.P.,University of Oxford | Cai Y.,VGXI Inc. | Hebel H.L.,VGXI Inc. | Hyde S.C.,University of Oxford | Gill D.R.,University of Oxford
Biomaterials | Year: 2011

Non-viral gene transfer using plasmid DNA (pDNA) is generally acknowledged as safe and non-immunogenic compared with the use of viral vectors. However, pre-clinical and clinical studies have shown that non-viral (lipoplex) gene transfer to the lung can provoke a mild, acute inflammatory response, which is thought to be, partly, due to unmethylated CG dinucleotides (CpGs) present in the pDNA sequence. Using a murine model of lung gene transfer, bronchoalveolar lavage fluid was collected following plasmid delivery and a range of inflammatory markers was analysed. The results showed that a Th1-related inflammatory cytokine response was present that was substantially reduced, though not abolished, by using CpG-free pDNA. The remaining minor level of inflammation was dependent on the quality of the pDNA preparation, specifically the quantity of contaminating bacterial genomic DNA, also a source of CpGs. Successful modification of a scalable plasmid manufacturing process, suitable for the production of clinical grade pDNA, produced highly pure plasmid preparations with reduced genomic DNA contamination. These studies help define the acceptable limit of genomic DNA contamination that will impact FDA/EMEA regulatory guidelines defining clinical grade purity of plasmid DNA for human use in gene therapy and vaccination studies. © 2011 Elsevier Ltd.

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